Uridine phosphorylase (UPase) inhibitors for treatment of liver conditions

ABSTRACT

Methods of treating a subject for a liver condition, e.g., NAFLD, NASH, and/or DILI, are provided. Aspects of the methods include administering to the subject an effective amount of a UPase inhibitor, optionally in combination with a uridine active agent (e.g., uridine (UR), a UR pro-drug or a UR mimetic), such as supplemental uridine, to treat the subject for the liver condition. Also provided are compositions for use in practicing the subject methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. § 119(e), this application claims priority to thefiling date of U.S. Provisional Patent Application Ser. No. 62/864,695filed Jun. 21, 2019; the disclosure of which application is hereinincorporated by reference.

INTRODUCTION

The prevalence of nonalcoholic fatty liver disease (NAFLD) andnonalcoholic steatohepatitis (NASH), a more severe form of NAFLD, isincreasing rapidly worldwide.¹ NAFLD features hepatic fat accumulation(steatosis), occasional fibrosis, and hepatocyte ballooning as theresult of accumulation of fat (triglyceride) droplets with no othercause of secondary hepatic fat accumulation (e.g., alcohol, infections,medications, etc.).² In addition to hepatocyte ballooning and fataccumulation, like found in NALFD, NASH also features lobularinflammation, fibrosis, and hepatocyte degeneration. For NASH, thefibrosis is typically followed by cirrhosis and end-stage liver disease,which is typically fatal without a liver transplant.³ In addition toend-stage liver disease, individuals with NASH often also develop livercancer (hepatocellular cancer, HCC) as the result of thecondition.^(4,5) ¹ Jennings J, Faselis C, Yao M D. NAFLD-NASH: AnUnder-Recognized Epidemic. Curr Vasc Pharmacol. 018; 16:209-213.² Koch LK, Yeh M M. Nonalcoholic fatty liver disease (NAFLD): Diagnosis,pitfalls, and staging. Ann Diagn Pathol. 2018; 37:83-90.³ Alkhouri N,Lawitz E, Noureddin M. Looking Into the Crystal Ball: Predicting theFuture Challenges of Fibrotic NASH Treatment. Hepatol Commun. 2019;3:605-613.⁴ Anstee Q M, Reeves H L, Kotsiliti E, Govaere O, HeikenwalderM. From NASH to HCC: current concepts and future challenges. Nat RevGastroenterol Hepatol. 2019 Apr. 26.⁵ Dhanasekaran R, Felsher D W. ATale of Two Complications of Obesity: Nonalcoholic steatohepatitis(NASH) and Hepatocellular carcinoma (HCC). Hepatology. 2019 Apr. 8.

The increased prevalence of both NALFD and NASH mirrors the societalincrease in obesity and type 2 diabetes (T2D), and reflects the hepaticmanifestation of the resulting altered metabolic state.⁶ The exposure ofhepatocytes to high concentrations of lipids and carbohydrates, termedlipotoxicity and glucotoxicity, respectively, underlie much of thehepatocellular injury observed in NAFLD/NASH.⁷ Metabolic syndrome,defined as a constellation of obesity, insulin resistance,hyperglycemia, dyslipidemia and hypertension, is the major risk factorpredisposing individuals to NAFLD and NASH.⁸ Approximately 30% of NorthAmericans suffer from NALFD and 4% from NASH.⁹ Genetic, demographic, andethnic factors may also play a role in the pathogenesis of NAFLD,¹⁰ forexample:

-   -   NAFLD has been linked with various genetic variants, including        PNPLA-3, TM6SF2, and FDFT1.    -   NAFLD is more common in older age groups and in men.    -   Hispanics have the highest prevalence of NAFLD in the US,        followed by Caucasians and then African Americans. ⁶ Esler W P,        Bence K K. Metabolic Targets in Nonalcoholic Fatty Liver        Disease. Cell Mol Gastroenterol Hepatol. 2019 Apr. 18.⁷ Mota M,        Banini B A, Cazanave S C, Sanyal A J, Molecular mechanisms of        lipotoxicity and glucotoxicity in nonalcoholic fatty liver        disease. Metabolism. 2016, 65:1049-61.⁸ Aguilar-Salinas Calif.,        Viveros-Ruiz T. Recent advances in managing/understanding the        metabolic syndrome. F1000Res. 2019 Apr. 3; 8.⁹ Garber K. The new        liver epidemic. Nat Biotechnol. 2019; 37:209-214.¹⁰ Iqbal U,        Perumpail B J, Akhtar D, Kim D, Ahmed A. The Epidemiology, Risk        Profiling and Diagnostic Challenges of Nonalcoholic Fatty Liver        Disease. Medicines (Basel). 2019; 6(1).

There are no US Food and Drug Administration (FDA) approved medicationsto treat patients with NASH. Current recommended actions to treat thedisease include weight loss and dietary modifications, e.g., loweringconsumption of fats and glucose.¹¹ The only known “cure” for late-stageNASH and/or HCC is a liver transplant. In this regard, NASH is set tosoon overtake hepatitis as the single leading factor leading to livertransplantation.⁹ ¹¹ Huang M A, Greenson J K, Chao C, Anderson L,Peterman D, Jacobson J, Emick D, Lok A S, Conjeevaram H S. One-yearintense nutritional counseling results in histological improvement inpatients with non-alcoholic steatohepatitis: a pilot study. Am JGastroenterol. 2005; 100:1072-81.

Lipid Droplets (LD), also referred to as lipid bodies, oil bodies, oradiposomes are dynamic organelles that store neutral lipids duringperiods of energy excess, and serve as an energy reservoir duringdeprivation.¹² Many prevalent metabolic diseases, such as metabolicsyndrome and obesity, often result in elevated lipids and increased LDsin the liver, also called hepatic steatosis/NAFLD. LD, and particularlyproteins associated with LD, are strongly associated with thepathophysiology of fatty liver disease.¹³ Under normal physiologicconditions, hepatic LDs are small and present in limited numbers. Moresubstantial LD formation is associated with hepatotoxicity postadministration to animals or man of drugs such as tamoxifen(TAM),^(14,15,16,17) cyclosporin,¹⁸ valproic acid,^(19,20)tetracyclines,²¹ clofibrate,²² olanzapine,²³ and statins such assimavastin,²⁴ etc. LDs appear to have a close relationship withmitochondria, including physical contact and protein shuttling.²⁵ LDsalso appear to have a similar intimate relationship with the cellnucleus.²⁶ The content of the LD appears to determine its potential fortoxicity-LDs containing unsaturated fatty acids like arachidonic acid,the precursor to many inflammatory mediators,²⁷ are more toxic than LDscontaining saturated fatty acids.²⁸ LDs are coated with proteins fromthe perilipin family, some of which are involved in the regulation oflipid metabolism.²⁹ ¹² Meyers A1, Weiskittel TM1, Dalhaimer P2,3. LipidDroplets: Formation to Breakdown. Lipids. 2017: May 20 [Epub ahead ofprint]¹³ Okumura T, Role of lipid droplet proteins in liver steatosis. JPhysiol Biochem. 2011; 67: 629-36.¹⁴ Nishino M, Hayakawa K, Nakamura Y,Morimoto T, Mukaihara S. Effects of tamoxifen on hepatic fat content andthe development of hepatic steatosis in patients with breast cancer:high frequency of involvement and rapid reversal after completion oftamoxifen therapy. AJR Am J Roentgenol 2003; 180: 129-134.¹⁵ Ogawa Y,Murata Y, Nishioka A, Inomata T, Yoshida S, Tamoxifen-induced fattyliver in patients with breast cancer. Lancet 1998; 351(9104):725.¹⁶Nguyen M C, Stewart R B, Banerji M A, Gordon D H, Kral J G.Relationships between tamoxifen use, liver fat and body fat distributionin women with breast cancer. Int J Obes Relat Metab Disord 2001; 25(2):296-298.¹⁷ Lüllmann H, Lüllmann-Rauch R. Tamoxifen-induced generalizedlipidosis in rats subchronically treated with high doses. Toxicol. Appl.Pharmacol. 1981; 61: 138-146.¹⁸ Wolf A, Trendelenburg C F,Diez-Fernandez C, Prieto P, Houy S, Trommer W E, Cordier A. CyclosporineA-induced oxidative stress in rat hepatocytes. J. Pharmacol. Exp. Ther.1996; 280: 1328-1334.¹⁹ Tong V, Teng X W, Chang T K, Abbott F S.Valproic acid I: time course of lipid peroxidation biomarkers, livertoxicity, and valproic acid metabolite levels in rats. Toxicol. Sci.2005; 86: 427-435.²⁰ Tong V, Teng X W, Chang T K, Abbott F S. 2005b.Valproic acid II: effects on oxidative stress, mitochondrial membranepotential, and cytotoxicity in glutathione-depleted rat hepatocytes.Toxicol. Sci. 2005; 86: 436-443.²¹ Amacher D E, Martin B A.Tetracycline-induced steatosis in primary canine hepatocyte cultures.Fundam. Appl. Toxicol. 1997; 40: 256-263.²² Meijer J, Afzelius B A.Effects of clofibrate treatment and of starvation on peroxisomes,mitochondria, and lipid droplets in mouse hepatocytes: a morphometricstudy. J Ultrastruct Mol Struct Res. 1989; 102: 87-94.²³ Nimura S,Yamaguchi T, Ueda K, Kadokura K, Aiuchi T, Kato R, Obama T, Itabe H.Olanzapine promotes the accumulation of lipid droplets and theexpression of multiple perilipins in human adipocytes. Biochem BiophysRes Commun. 2015; 467: 906-12.²⁴ Gbelcová H, Svéda M, Laubertová L,Varga I, Vítek L, Kolář M, Strnad H, Zelenka J, Böhmer D, Ruml T. Theeffect of simvastatin on lipid droplets accumulation in human embryonickidney cells and pancreatic cancer cells. Lipids Health Dis. 2013;12:126.²⁵ Bischof J, Salzmann M, Streubel M K, Hasek J, Geltinger F,Duschl J, Bresgen N, Briza P, Haskova D, Lejskova R, Sopjani M, RichterK, Rinnerthaler M. Clearing the outer mitochondrial membrane fromharmful proteins via lipid droplets. Cell Death Discov. 2017; 3:17016.²⁶Welte M A. Expanding roles for lipid droplets. Curr Biol. 2015; 25:R470-81.²⁷ Martin S A, Brash A R, Murphy R C. The discovery and earlystructural studies of arachidonic acid. J Lipid Res. 2016; 57:1126-32.²⁸ Czamara K, Majzner K, Selmi A, Baranska M, Ozaki Y, Kaczor A.Unsaturated lipid bodies as a hallmark of inflammation studied by Raman2D and 3D microscopy. Sci Rep. 2017; January 18; 7:40889.²⁹ Itabe H,Yamaguchi T, Nimura S, Sasabe N. Perilipins: a diversity ofintracellular lipid droplet proteins. Lipids Health Dis. 2017; 16: 83.

A major liver-related public health problem is drug induced liverdisease (DILI). affecting individuals consuming pharmaceutical ornutraceuticals. DILI has been linked to over 1000 drugs.³⁰ Althoughcomplete recovery is expected for patients experiencing less seriousDILI, the associated symptoms (e.g., fatigue, itching, nausea) can bedebilitating and recovery can be prolonged, with about 20% of patientshaving biochemical evidence of continuing liver injury 6 months afterdiagnosis.³¹ Cirrhosis and long-term liver-related morbidity andmortality occur in approximately 3% of cases.³² Currently no tests areavailable to physicians to confidently diagnosis DILI.³³ A DILI, or eventhe suspicion of a DILI, may lead to use of an alternate treatment,resulting in exposure to new adverse drug event risks and possiblesuboptimal treatment of the underlying disease. DILIs also are a commoncause for termination of clinical drug development programs.³⁴ DILIresults in a range of pathologies. These pathologies range fromelevation of serum transaminase levels, detected on routine, biochemicallaboratory testing, that resolves after removal of the offendingchemical agent to acute liver failure, defined as de novo, sudden, andlife-threatening liver dysfunction that leads to coagulopathy andhepatic encephalopathy within 26 weeks of the onset of illness.³⁵ Acuteliver failure is a devastating disease since it primarily affects young,healthy individuals and leads to death in approximately 30% of patientsreceiving aggressive therapy, including liver transplant. ³⁰ Iorga A,Dara L, Kaplowitz N. Drug-Induced Liver Injury: Cascade of EventsLeading to Cell Death, Apoptosis or Necrosis. Int J Mol Sci. 2017;18(5). E1018.³¹ Fontana, R. J. et al. Idiosyncratic drug-induced liverinjury is associated with substantial morbidity and mortality within 6months from onset. Gastroenterology. 2014; 147, 96-108.³²Saithanyamurthi H, Faust A J. Drug-Induced Liver Disease: ClinicalCourse. Clin Liver Dis. 2017; 21: 21-34.³³ Mosedale M, Watkins P B.Drug-induced liver injury: Advances in mechanistic understanding thatwill inform risk management. Clin Pharmacol Ther. 2017; 101: 469-480.³⁴Watkins P B, Drug safety sciences and the bottleneck in drugdevelopment. Clin Pharmacol Ther 2011; 89: 788-790.³⁵ Fisher K,Vuppalanchi R, Saxena R. Drug-Induced Liver Injury. Arch Pathol Lab Med.2015; 139: 876-87

There are two types of DILI, “intrinsic” and “idiosyncratic.”

-   -   Drugs that induce liver injury in a predictable, dose-dependent        manner in both preclinical models and humans are said to cause        intrinsic DILI. Acetaminophen is the most common cause of        intrinsic DILI in the US. Few other drugs on the market cause        life-threatening, intrinsic DILI because this liability is        generally identified during preclinical or early clinical        studies. Such drugs are often abandoned from further        development, used at doses below that providing optimum efficacy        but anticipated to not cause liver injury, or administered in        controlled or desperate situations, eg, chemotherapy. Except for        N-acetyl cysteine to treat acetaminophen overdose, there is no        treatment available for intrinsic DILIs, and N-acetyl cysteine        is useful in only limited clinical circumstances.    -   Idiosyncratic DILI is the most problematic form of DILI. It        occurs infrequently among treated patients, often after several        months of treatment with the offending drug. Idiosyncratic DILI        for a new drug is often only discovered when the drug enters        general use. Idiosyncratic DILI with latency likely reflects an        immune attack on the liver. Consistent with this view,        idiosyncratic liver injury will recur promptly after complete        recovery if the DILI patient is re-challenged with the offending        drug. The prolonged latency can be attributed to the time        required for antigen-specific lymphocytes to be activated and        proliferate to enough numbers to mediate the DILI. The likely        first step in initiating idiosyncratic DILI is formation of a        hepatocyte stress inducing neoantigen.

Both intrinsic and idiosyncratic liver damage appears to proceed throughsimilar processes. Proposed mechanisms include mitochondrialdysfunction, oxidative stress, and alterations in bile acid homeostasis.Mitochondria produce ATP that is required to maintain all vital cellularfunctions. DILI-causing drugs can inhibit mitochondrial function,resulting in reduced levels of ATP, a decline in cell function, andeventually cell death.³⁶ Oxidative stress is the result of ROS that area byproduct of normal metabolism and have roles in cell signaling andhomeostasis. However, some DILI-causing drugs can increase ROSaccumulation through a variety of mechanisms.³⁷ When the processes thatexist to regulate cellular levels of ROS are exceeded, oxidative stresscan result in damage to key cellular components and eventually celldeath. Finally, a major function of the liver is the transport of bilesalts from blood into bile. DILI-causing drugs can disrupt this processin many ways, most importantly through reducing hepatic bile acid effluxby inhibition of the bile salt export protein.³⁸ This results in theintracellular accumulation of toxic bile acids which can lead tohepatocyte death. In summary, DILI appears in two different variants,intrinsic or idiosyncratic, with similar pathology and pathogenesis, butlikely with multiple mechanisms of initiation and promotion. ³⁶ Will, Y.& Dykens, J. Mitochondrial toxicity assessment in industry—a decade oftechnology development and insight, Expert Opin Drug Metab Toxicol.2014; 10:1061-1067.³⁷ Gomez-Lechon, M J, Tolosa, L, Donato, M T.Metabolic activation and drug-induced liver injury: in vitro approachesfor the safety risk assessment of new drugs. J Appl Toxicol. 2016; 36:752-768.³⁸ Morgan R E, van Staden C J, Chen Y, Kalyanaraman N, KalanziJ, Dunn R T 2nd, Afshari C A, Hamadeh H K. A multifactorial approach tohepatobiliary transporter assessment enables improved therapeuticcompound development. Toxicol Sci. 2013; 136: 216-241.

The link between NAFLD/NASH and DILI in a still not completelyunderstood.^(39,40,41,42,43,44) However, (1) DILI is a risk factor inNAFLD/NASH patients for many drugs, (2) DILI presents as lesions thatresemble those of NAFLD/NASH, (3) the pathophysiology of DILI and NASHoverlap, (4) certain drugs induce hepatic steatosis (DIS) and/orsteatohepatitis (DISH) by triggering pathological events similar tothose occurring in the development and progression NAFLD/NASH, (5) DILIinfluences the development or accelerates progression of NAFLD/NASH, and(6) NAFLD/NASH affects the susceptibility to, and outcome of, DILI. ³⁹Massart J, Begriche K, Moreau C, Fromenty B. Role of nonalcoholic fattyliver disease as risk factor for drug-induced hepatotoxicity. J ClinTransl Res. 2017; 3 (Suppl 1):212-232⁴⁰ Ortega-Alonso A, Andrade R J.Chronic liver injury induced by drugs and toxins. J Dig Dis. 2018;19:514-521.⁴¹ Spinnenhirn V, Demgenski J, Brunner T. Death ReceptorInteractions With the Mitochondrial Cell Death Pathway During ImmuneCell-, Drug- and Toxin-Induced Liver Damage. Front Cell Dev Biol. 2019;7:72.⁴² Pavlik L, Regev A, Ardayfio P A, Chalasani N P. Drug-InducedSteatosis and Steatohepatitis: The Search for Novel Serum BiomarkersAmong Potential Biomarkers for Non-Alcoholic Fatty Liver Disease andNon-Alcoholic Steatohepatitis. Drug Saf. 2019 June; 42:701-711.⁴³ RegevA, Palmer M, Avigan M I, Dimick-Santos L, Treem W R, Marcinak J F,Seekins D, Krishna G, Anania F A, Freston J W, Lewis J H, Sanyal A J,Chalasani N. Consensus: guidelines: best practices for detection,assessment and management of suspected acute drug-induced liver injuryduring clinical trials in patients with nonalcoholic steatohepatitis.Aliment Pharmacol Ther. 2019; 49:702-713.⁴⁴ Massart J, Begriche K,Moreau C, Fromenty B. Role of nonalcoholic fatty liver disease as riskfactor for drug-induced hepatotoxicity. J Clin Transl Res. 2017; 3(Suppl1):212-232.

Harmful effects from DILI not only can mimic the physiologic insultsthat trigger the development and/or progression of NAFLD in the normalliver but also aggravate similar alterations pre-existing in a fattyliver. Relative to NAFLD/NASH, DILI:

-   -   Exacerbates predisposing factors (e.g., obesity, diabetes)    -   Enhances steatotic factors (eg, exacerbates lipid hepatic        synthesis/uptake)    -   Increases Inflammatory factors (eg, accumulation of lipotoxic        fatty acids and oxidative stress)    -   Activates fibrogenic factors (eg, enhanced collagen deposition)    -   Alters drug-metabolic systems that occur in the NAFLD context        Based on the above, an agent that mitigates NAFLD/NASH likely        would also act to treat DILI and vice-versa.

NAFLD/NASH has become a significant, potentially lethal, health epidemicin countries suffering increased prevalence of obesity and theassociated metabolic disorders for which there are no approvedtreatments. DILI, another serious hepatic condition appears to mimic thepathophysiology of NAFLD/NASH.

SUMMARY

Methods of treating a subject for a liver condition, e.g., NAFLD, NASH,and/or DILI, are provided. Aspects of the methods include administeringto the subject an effective amount of a UPase inhibitor, optionally incombination with a uridine active agent (e.g., uridine (UR), a URpro-drug or a UR mimetic), such as supplemental uridine, to treat thesubject for the liver condition. Also provided are compositions for usein practicing the subject methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a regression analysis of plasma UR concentration versusplasma Compound 1 concentrations determined following continuousinfusion of various amounts of Compound 1 (TK-112690) to mice. R2 forthe line is 0.95, and the slope and intercept values for the line are0.010 and 0.051, respectively. Compound 1 is seen to elevate plasma URin a linear fashion.

FIG. 2 provides the body weights of the mice measured during theexperimental phase of the MCD study. Six mice per experimental groupwere studied. All groups showed substantial weight loss. No variation inbody weight was observed between the groups. Data presented asmean+/−SEM.

FIG. 3 provides serum HDL cholesterol levels measured at the end of theexperimental phase of the MCD study. Six mice per experimental groupwere studied. Mice treated with UR+Compound 1 (TK-112690) hadsignificantly lower (p<0.05) HDL cholesterol levels compared to vehicletreated controls. Data presented as mean+/−SEM.

FIG. 4 provides (A) representative H&E images of liver sections fromeach experimental group and (B) fibrosis scoring of the images shown in(A). The greatest effect was the group of mice treated with UR+Compound1 (TK-112690). This group displayed significantly (p<0.001) less liverfibrosis compared to vehicle treated controls (six animals perexperimental group). Data presented as mean+/−SEM.

DEFINITIONS

The following terms have the following meanings unless otherwiseindicated when describing the compounds, pharmaceutical compositionscontaining such compounds, methods of using such compounds andcompositions, and the description of the biology and pharmacology foruse of the compounds, It should also be understood that any of themoieties defined forth below may be substituted with a variety ofsubstituents, and that the respective definitions are intended toinclude such substituted moieties within their scope.

“Acyl” refers to a radical —C(O)R, where R is hydrogen, alkyl,cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroalkyl, orheteroaryl as defined herein. Representative examples include, but arenot limited to, formyl, acetyl, cyclohexylcarbonyl,cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl and the like.

“Acylamino” refers to a radical —NR′C(O)R, where R′ is hydrogen, alkyl,cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl,heteroarylalkyl and R is hydrogen, alkyl, alkoxy, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl orheteroarylalkyl, as defined herein. Representative examples include, butare not limited to, formylamino, acetylamino, cyclohexylcarbonylamino,cyclohexylmethyl-carbonylamino, benzoylamino, benzylcarbonylamino andthe like.

“Acyloxy” refers to the group —OC(O)H, —OC(O)-alkyl, —OC(O)-aryl or—OC(O)— cycloalkyl.

“Aliphatic” refers to hydrocarbyl organic compounds or groupscharacterized by a straight, branched or cyclic arrangement of theconstituent carbon atoms and an absence of aromatic unsaturation.Aliphatics include, without limitation, alkyl, alkylene, alkenyl,alkynyl and alkynylene. Aliphatic groups typically have from 1 or 2 to 6or 12 carbon atoms.

“Alkenyl” refers to monovalent olefinically unsaturated hydrocarbylgroups having up to about 11 carbon atoms, particularly, from 2 to 8carbon atoms, and more particularly, from 2 to 6 carbon atoms, which canbe straight-chained or branched and having at least 1 and particularlyfrom 1 to 2 sites of olefinic unsaturation. Particular alkenyl groupsinclude ethenyl (—CH═CH₂), n-propenyl (—CH₂CH═CH₂), isopropenyl(—C(CH₃)═CH₂), vinyl and substituted vinyl, and the like.

“Alkoxy” refers to the group —O-alkyl. Particular alkoxy groups include,by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy,tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, andthe like.

“Alkoxycarbonyl” refers to a radical —C(O)-alkoxy where alkoxy is asdefined herein.

“Alkoxycarbonylamino” refers to the group —NRC(O)OR′ where R ishydrogen, alkyl, aryl or cycloalkyl, and R′ is alkyl or cycloalkyl.

“Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groupsparticularly having up to about 12 or 18 carbon atoms, more particularlyas a lower alkyl, from 1 to 8 carbon atoms and still more particularly,from 1 to 6 carbon atoms. The hydrocarbon chain may be eitherstraight-chained or branched. This term is exemplified by groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, tert-butyl,n-hexyl, n-octyl, tert-octyl and the like. The term “alkyl” alsoincludes “cycloalkyls” as defined herein. Structures for a few exemplaryalkyl groups are provided in Table 1 below.

TABLE 1 Structure of exemplary alkyl groups

“Alkylene” refers to divalent saturated aliphatic hydrocarbyl groupsparticularly having up to about 12 or 18 carbon atoms and moreparticularly 1 to 6 carbon atoms which can be straight-chained orbranched. This term is exemplified by groups such as methylene (—CH₂—),ethylene (—CH₂CH₂—), the propylene isomers (eg, —CH₂CH₂CH₂— and —CH(CH₃)CH₂—) and the like.

“Alkynyl” refers to acetylenically unsaturated hydrocarbyl groupsparticularly having up to about 12 or 18 carbon atoms and moreparticularly 2 to 6 carbon atoms which can be straight-chained orbranched and having at least 1 and particularly from 1 to 2 sites ofalkynyl unsaturation. Particular non-limiting examples of alkynyl groupsinclude acetylenic, ethynyl (—C≡CH), propargyl (—CH₂C≡CH), and the like.

“Amino” refers to the radical —NH₂.

“Amino acid” refers to any of the naturally occurring amino acids (egAla, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu, Lys,Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D, L, or DL form. Theside chains of naturally occurring amino acids are well known in the artand include, for example, hydrogen (eg, as in glycine), alkyl (eg, as inalanine, valine, leucine, isoleucine, proline), substituted alkyl (eg,as in threonine, serine, methionine, cysteine, aspartic acid,asparagine, glutamic acid, glutamine, arginine, and lysine), alkaryl(eg, as in phenylalanine and tryptophan), substituted arylalkyl (eg, asin tyrosine), and heteroarylalkyl (eg, as in histidine).

“Aminocarbonyl” refers to the group —C(O)NRR where each R isindependently hydrogen, alkyl, aryl or cycloalkyl, or where the R groupsare joined to form an alkylene group.

“Aminocarbonylamino” refers to the group —NRC(O)NRR where each R isindependently hydrogen, alkyl, aryl or cycloalkyl, or where two R groupsare joined to form an alkylene group.

“Aminocarbonyloxy” refers to the group —OC(O)NRR where each R isindependently hydrogen, alkyl, aryl or cycloalky, or where the R groupsare joined to form an alkylene group.

“Amino-containing saccharide group” refers to a saccharide group havingan amino substituent. Representative amino-containing saccharide includeL-vancosamine, 3-desmethyl-vancosamine, 3-epi-vancosamine,4-epi-vancosamine, acosamine, actinosamine, daunosamine,3-epi-daunosamine, ristosamine, N-methyl-D-glucamine and the like.”

“ARMD” refers to an eye disease age related macular degeneration.

“Aralkyl” or “arylalkyl” refers to an alkyl group, as defined above,substituted with one or more aryl groups, as defined above.

“Aryl” refers to a monovalent aromatic hydrocarbon group derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Typical aryl groups include, but are not limitedto, groups derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene and the like. Particularly, anaryl group comprises from 6 to 14 carbon atoms. The structures of a fewexemplary aryl groups are provided in Table 2.

TABLE 2 Examples of aryl groups

“Aryloxy” refers to —O-aryl groups wherein “aryl” is as defined herein.

“Autoimmune disease” or “autoimmune condition” refers an illness thatoccurs when the body tissues are attacked by its own immune system.Examples of autoimmune disease or conditions include multiple sclerosis,ankylosing spondylitis, Crohn's disease, arthritis, psoriasis, Behçet'sdisease and psoriatic arthritis.

Azido” refers to the radical —N₃.

“Carbohydrate” means a mono-, di-, tri-, or polysaccharide, wherein thepolysaccharide can have a molecular weight of up to about 20,000, forexample, hydroxypropyl-methylcellulose or chitosan. “Carbohydrate” alsoencompasses oxidized, reduced or substituted saccharide monoradicalcovalently attached to the anhydropyrimidine (eg, anhydrothymidine oranhydrouridine), or derivative thereof any atom of the saccharidemoiety, eg, via the aglycone carbon atom. The “mono-, di-, tri-, orpolysaccharide” can also include amino-containing saccharide groups.Representative “carbohydrate” include, by way of illustration, hexosessuch as D-glucose, D-mannose, D-xylose, D-galactose, vancosamine,3-desmethyl-vancosamine, 3-epi-vancosamine, 4-epi-vancosamine,acosamine, actinosamine, daunosamine, 3-epi-daunosamine, ristosamine,D-glucamine, N-methyl-D-glucamine, D-glucuronic acid,N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, sialyic acid, iduronicacid, L-fucose, and the like; pentoses such as D-ribose or D-arabinose;ketoses such as D-ribulose or D-fructose; disaccharides such as2-O-(α-L-vancosaminyl)-β-D-glucopyranose-,2-O-(3-desmethyl-α-L-vancosaminyl)-β-D-glucopyranose, sucrose, lactose,or maltose; derivatives such as acetals, amines, acylated, sulfated andphosphorylated sugars; oligosaccharides having from 2 to 10 saccharideunits. The saccharides can be either in their open, r pyranose orfuranose forms.

“Carboxyl” refers to the radical —C(O)OH.

“Cyano” refers to the radical —CN.

“Cycloalkenyl” refers to cyclic hydrocarbyl groups having from 3 to 10carbon atoms and having a single cyclic ring or multiple condensedrings, including fused and bridged ring systems and having at least oneand particularly from 1 to 2 sites of olefinic unsaturation. Suchcycloalkenyl groups include, by way of example, single ring structuressuch as cyclohexenyl, cyclopentenyl, cyclopropenyl, and the like.

“Cycloalkyl” refers to cyclic hydrocarbyl groups having from 3 to about10 carbon atoms and having a single cyclic ring or multiple condensedrings, including fused and bridged ring systems, which optionally can besubstituted with from 1 to 3 alkyl groups. Such cycloalkyl groupsinclude, by way of example, single ring structures such as cyclopropyl,cyclobutyl, cyclopentyl, cyclooctyl, 1-methylcyclopropyl,2-methylcyclopentyl, 2-methylcyclooctyl, and the like, and multiple ringstructures such as adamantanyl, and the like.

“DILI” refers drug-induced liver injury

“DIS” refers drug-induced hepatic steatosis

“DISH” refers to drug-induced steatohepatitis.

“DR” refers to an eye condition, diabetic retinopathy.

“FU” refers to 5-fluorouracil,

“Heterocycloalkyl” refers to a stable heterocyclic non-aromatic ring andfused rings containing one or more heteroatoms independently selectedfrom N, O and S. A fused heterocyclic ring system may includecarbocyclic rings and need only include one heterocyclic ring. Examplesof heterocyclic rings include, but are not limited to, piperazinyl,homopiperazinyl, piperidinyl and morpholinyl. The structures of a fewexemplary heterocyclyls are shown in Table 3.

TABLE 3 Examples of heterocyclyls

“Halo” or “halogen” refers to fluoro, chloro, bromo and iodo. Halogroups can be either fluoro or chloro.

“HCC” refers to hepatic cell carcinoma

“HDL” refers to high density lipoprotein.

“Hetero” when used to describe a compound or a group present on acompound means that one or more carbon atoms in the compound or grouphave been replaced by a nitrogen, oxygen, or sulfur heteroatom. Heteromay be applied to any of the hydrocarbyl groups described above such asalkyl, eg heteroalkyl, cycloalkyl, eg heterocycloalkyl, aryl, egheteroaryl, cycloalkenyl, eg, heterocycloalkenyl, cycloheteroalkenyl,eg, heterocycloheteroalkenyl and the like having from 1 to 5, andparticularly from 1 to 3 heteroatoms. A heteroatom is any atom otherthan carbon or hydrogen and is typically, but not exclusively, nitrogen,oxygen, sulfur, phosphorus, boron, chlorine, bromine, or iodine. Anunsubstituted heteroatom refers to a pendant heteroatom such as anamine, hydroxyl and thiol. A substituted heteroatom refers to aheteroatom that is other than a pendant heteroatom.

“Heteroaryl” refers to a monovalent heteroaromatic group derived by theremoval of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Typical heteroaryl groups include, but arenot limited to, groups derived from acridine, arsindole, carbazole,β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole,indole, indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene, and the like. The heteroaryl group can bea 5-20 membered heteroaryl, or 5-10 membered heteroaryl. Particularheteroaryl groups are those derived from thiophen, pyrrole,benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole,oxazole and pyrazine.

“Hydroxyl” refers to the radical —OH.

“KO” refers to knockout as used in the phrase knockout animals.

“MCD” refers to methionine-choline deficient diet

“NAFLD” refers to non-alcoholic fatty liver disease.

“NASH” refers to non-alcoholic steatohepatitis

“Nitro” refers to the radical —NO₂.

“Peptide” refers to a polyamino acid containing up to 2, 5, 10, or about100 amino acid residues.

“Polypeptide” means polyamino acid containing from about 100 amino acidunits to about 1,000 amino acid units, from about 100 amino acid unitsto about 750 amino acid units, or from about 100 amino acid units toabout 500 amino acid units.

“ROP refers to an eye condition in infant's retinopathy of prematurity.

“SEM” refers to standard error of the mean

“Side-effect” means an undesirable adverse consequence of drugadministration.

“Stereoisomer” as it relates to a given compound is well understood inthe art, and refers to another compound having the same molecularformula, wherein the atoms making up the other compound differ in theway they are oriented in space, but wherein the atoms in the othercompound are like the atoms in the given compound with respect to whichatoms are joined to which other atoms (eg an enantiomer, a diastereomer,or a geometric isomer). For example, Morrison and Boyd, OrganicChemistry, 1983, 4th ed., Allyn and Bacon, Inc., Boston, Mass., p 123.

“Substituted” refers to a group in which one or more hydrogen atoms areeach independently replaced with the same or different substituent(s).“Substituted” groups particularly refer to groups having 1 or moresubstituents, for instance from 1 to 5 substituents, and particularlyfrom 1 to 3 substituents, selected from the group consisting of acyl,acylamino, acyloxy, alkoxy, substituted alkoxy, alkoxycarbonyl,alkoxycarbonylamino, amino, substituted amino, aminocarbonyl,aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, aralkyl, azido,carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen, hydroxyl,imidate, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy,thioketo, thiol, alkylthio, (substituted alkyl)thio, arylthio,(substituted aryl)thio, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)₂— andaryl-S(O)₂. Typical substituents include, but are not limited to, —X,—R⁸ (with the proviso that R⁸ is not hydrogen), —O—, ═O, —OR⁸, —SR⁸,—S—, ═S, —NR⁸R⁹, ═NR⁸, —CX₃, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃,—S(O)₂O⁻, —S(O)₂OH, —S(O)₂R⁸, —OS(O₂)O⁻, —OS(O)₂R⁸, —P(O)(O—)₂,—P(O)(OR⁸)(O⁻), —OP(O)(OR⁸)(OR⁹), —C(O)R⁸, —C(S)R⁸, —C(O)OR⁸,—C(O)NR⁸R⁹, —C(O)O⁻—, —C(S)OR⁸, —NR¹⁰C(O)NR⁸R⁹, —NR¹⁰C(S)NR⁸R⁹,—NR¹¹C(NR¹)NR⁸R⁹ and —C(NR)NR⁸R⁹, where X is independently a halogen.

“Substituted amino” includes those groups recited in the definition of“substituted” herein, and particularly refers to the group —N(R)₂ whereeach R is independently selected from the group consisting of hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, cycloalkyl, substituted cycloalkyl, and whereboth R groups are joined to form an alkylene group.

“T2D” refers to type 2 diabetes.

“TG” refers to transgenic

“Thioalkoxy” refers to the group —S-alkyl.

“Thioaryloxy” refers to the group —S-aryl.

“Thioketo” refers to the group ═S.

“Thiol” refers to the group —SH.

“UR” refers to uridine.

“UPase (Uridine phosphorylase)” refers in enzymology to a phosphorylase(EC 2.4.2.3) that catalyzes the chemical reaction:uridine+phosphate→uracil+alpha-D-ribose 1-phosphate. The two substratesof this enzyme are uridine and phosphate, whereas its two products areuracil and alpha-D-ribose 1-phosphate. This enzyme belongs to the familyof glycosyltransferases, specifically the pentosyltransferases. Thesystematic name of this enzyme class is uridine: phosphatealpha-D-ribosyltransferase. Other names in common use include pyrimidinephosphorylase, UrdPase, UPH, and UPase. This enzyme participates inpyrimidine metabolism.

“Uridine Supplement” refers to either a formulated product containing URor a formulated product containing a UR precursor such as URmonophosphate or acetylated UR that converts to UR in the body. Theformulated product could be a solution, a capsule, a tablet or a cream.The product could be administered po, ip, sc, or iv. The UR supplementcould be administered as part of a more complex mixture such as anutritional supplement.

ip, po and sc are intraperitoneal, oral or subcutaneous dosing,respectfully. H&E is Haematoxylin & Eosin, a dye used to stain tissues.SD is standard deviation. SE is standard error. PBS is phosphatebuffered saline. qd. and bid are daily and twice-a-day, respectfully.

One having ordinary skill in the art will recognize that the maximumnumber of heteroatoms in a stable, chemically feasible heterocyclicring, whether it is aromatic or non-aromatic, is determined by the sizeof the ring, the degree of unsaturation and the valence of theheteroatoms. In general, a heterocyclic ring may have one to fourheteroatoms so long as the heteroaromatic ring is chemically feasibleand stable.

DETAILED DESCRIPTION

Methods of treating a subject for a liver condition, e.g., NAFLD, NASH,and/or DILI, are provided. Aspects of the methods include administeringto the subject an effective amount of a UPase inhibitor, optionally incombination with a uridine active agent (e.g., uridine (UR), a URpro-drug or a UR mimetic), such as supplemental uridine, to treat thesubject for the liver condition. Also provided are compositions for usein practicing the subject methods.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

While the apparatus and method has or will be described for the sake ofgrammatical fluidity with functional explanations, it is to be expresslyunderstood that the claims, unless expressly formulated under 35 U.S.C.§ 112, are not to be construed as necessarily limited in any way by theconstruction of “means” or “steps” limitations, but are to be accordedthe full scope of the meaning and equivalents of the definition providedby the claims under the judicial doctrine of equivalents, and in thecase where the claims are expressly formulated under 35 U.S.C. § 112 areto be accorded full statutory equivalents under 35 U.S.C. § 112.

In further describing the subject invention, the subject methods aredescribed first in greater detail, followed by a review of the variouscompositions, e.g., formulations and kits, that may find use in thesubject methods, as well as a discussion of various representativeapplications in which the subject methods and compositions find use.

Methods

As summarized above, methods of treating, including prophylacticallytreating (e.g., preventing the occurrence of) a liver condition in asubject are provided. Aspects of the methods include administration tothe subject of a UPase inhibitor, either alone or in combination with auridine (UR) active agent (e.g., UR, a UR pro-drug or UR mimetic), totreat the subject for the liver condition. Where the UPase inhibitor isadministered in combination, i.e., concurrently, with the UR activeagent, the UPase inhibitor may be administered simultaneously with theUPase inhibitor. Alternatively, the UPase inhibitor and UR active agentmay be administered sequentially, e.g., where the UPase inhibitor isadministered before the UR active agent or the UPase in inhibitor isadministered after the UR active agent. In such embodiments, the UPaseinhibitor and the UR active agent can be administered at the same time,e.g., as two separate formulations, or combined into a singlecomposition. Alternately, the UPase inhibitor and the UR active agentcan be administered sequentially to the subject in differentformulations. Regardless of whether the UPase inhibitor and the URactive agent are administered sequentially or simultaneously, or anyeffective variation thereof, the agents are considered to beadministered together or in combination for purposes of the presentinvention. Routes of administration of the two agents may vary.Representative routes of administration are described below.

Subjects that are treated according to methods of the invention may besubjects suffering from, or suspected to suffer from, a liver condition,such as NAFLD, NASH or DILI. Treatment according to the disclosedmethods can begin prophylactically for subjects at risk for liverdisease or post diagnosis of a serious liver condition. Treatment can becarried out at intervals determined to be appropriate by those of skillin the art. For example, the administration can be carried out 1, 2, 3,4 or more times/day. Ideally, treatment is expected to be qdchronically. Treatment can also be started before or at or near the sametime as a drug associated with serious liver conditions.

The dose administered to an animal, particularly a human, in the contextof the present invention should be sufficient to affect a prophylacticor therapeutic response in the animal over a reasonable time frame. Oneskilled in the art will recognize that dosage will depend on a varietyof factors including the strength of the particular compound employedand the dosing regimen used, the condition of the animal, and the bodyweight of the animal, as well as the severity of the illness and thestage of the disease. The size of the dose will also be determined bythe existence, nature, and extent of any adverse side-effects that mightaccompany the administration of a particular compound.

UPase Inhibitor

As summarized above, aspects of the invention include administration tothe subject of a UPase inhibitor. UPase (UPh; EC 2.4.2.3) is a member ofthe pyrimidine nucleoside phosphorylase family of enzymes whichcatalyzes the phosphorolytic cleavage of the C—N glycoside bond of UR,with the formation of ribose 1-phosphate and uracil.⁴⁵ The UPaseinhibitor is an agent that acts to modulate uridine plasma level in thesubject, e.g., an agent that acts to elevate uridine (UR) plasma levelin the subject. While the magnitude of any UR plasma level enhancementmay vary, in some instances the magnitude of enhancement is 2-fold orgreater, such as 5-fold or greater, 10-fold or greater, 15-fold orgreater, 20-fold or greater, 25-fold or greater, or 50-fold or greater.⁴⁵ Pizzorno G1, Cao D, Leffert J J, Russell R L, Zhang D, HandschumacherR E. Homeostatic control of uridine and the role of uridinephosphorylase: a biological and clinical update. Biochim Biophys Acta.2002; 1587(2-3):133-44.

In some instances, the UPase inhibitor is an anhydronucleoside.Anhydronucleosides are analogs of natural nucleosides, often finding useas intermediates in the synthesis of nucleoside derivatives. They arecharacterized by having, in addition to the N-glycoside linkage, acovalent linkage either directly or via bridging atoms between the 2′,3′, or 5′ carbons of the sugar and a carbon, oxygen or nitrogen atom(other than the nitrogen of the glycoside bond) of the base. Theanhydropyrimidines are characterized by a pyrimidine base that iscovalently linked either directly or via bridging atoms between the 2′,3′, or 5′ carbons of the sugar and a carbon, oxygen or nitrogen atom(other than the nitrogen of the glycoside bond) of the pyrimidine base.

In some instances, the UPase inhibitor is a 2,2′-anhydropyrimidine orderivative thereof. In some embodiments, the 2,2′-anhydropyrimidine orderivative thereof is a compound of formula (I):

or the pharmaceutically acceptable salts, solvates, hydrates, andprodrug forms thereof, and stereoisomers thereof;

wherein:

each R¹, R², R³ and R⁴ is independently selected from the groupconsisting of hydrogen, substituted or unsubstituted heteroatom,substituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,substituted or unsubstituted aralkyl, hydroxyl, halogen, azido, amino,substituted amino, carbohydrate, nucleic acid, amino acid, peptide, dye,fluorophore and polypeptide.

In certain embodiments, the compound is of formula (I), R¹, R², R³ andR⁴ are independently hydrogen, hydroxyl, heteroatom, C₁-C₁₈ alkyl,C₁-C₁₈ substituted alkyl, C₁-C₁₈ alkenyl, C₁-C₁₈ acyl, amino,substituted amino, wherein the alkyl, alkenyl or acyl is linear orbranched, and optionally substituted with a hydroxyl, an ester and itsderivatives, a carboxyl and its derivatives, a cycloalkyl, aheterocycloalkyl, an aryl, a heteroaryl, an aralkyl, a heteroatom, andpossibly containing in chain or bridging heteroatoms such as nitrogen,oxygen and sulfur.

Examples of R¹ constituents of interest include, but are not limited to:hydrogen; hydroxyl; sulfhydryl; halogen such as fluorine, chlorine,bromine or iodine, as well as pseudohalogen such as a loweralkylsulfonyl group of 1 to 5 carbons such as methyl-, ethyl-, propyl-,isopropyl-, butyl-, isobutyl-, tert-butyl-, and pentasulfonyl orarylsulfonyl such as benzene, p-toluene, p-nitrobenzenesulfonyl groups;lower alkyl containing 1 to 20 carbons such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl and the like, includingsubstituted lower alkyl such as aminomethyl, hydroxymethyl, methoxy,ethyloxy, propyloxy, benzyloxy, imidate, alkylthio, (substitutedalkyl)thio, arylthio, (substituted aryl)thio and the like; lower alkenylcontaining 1 to 20 carbons such as vinyl and substituted vinyl, ethynyland substituted ethynyl, where the substituted vinyl or substitutedethynyl designates substitution of the β position of vinyl or ethynyl bya halogen such as bromine, chlorine, fluorine or iodine, or substitutionby an alkyl of 1 to 5 carbon atoms such as methyl, ethyl, propyl, butyl,pentyl and the like, or aralkyl such as benzyl, p-chlorobenzyl,p-nitrobenzyl and the like, or aryl such as phenyl, p-nitrophenyl,p-tolyl, p-anisyl, naphtyl and the like; lower alkanoyl (acyl groups)containing 1 to 20 carbons such as formyl, acetyl, propionyl,isopropionyl, butyryl, isobutyryl, tert-butyryl, valeryl, pivaloyl,caproyl, capryl, lauryl, myristyl, palmityl, stearyl, arachidyl,stilligyl, palmitoyl, oleyl, linolenyl, arachidonoyl and the like; loweraryl containing 1 to 20 carbons such as phenyl, p-tolyl, p-chlorophenyl,p-aminophenyl, p-nitrophenyl, p-anisyl and the like; lower aroylcontaining 1 to 20 carbons such as benzoyl and naphthoyl, where thearomatic group may be additionally substituted by alkyl, alkoxy, halo,or nitro moieties such as p-tolnoyl, p-anisoyl, p-chlorobenzoyl,p-nitrobenzoyl or 2,4-dinitrobenzoyl, pentafluorobenzoyl and the like,or another aroyl such as benzyloxybenzyl and the like; lower aralkylcontaining 1 to 20 carbons such as benzyl, benzhydryl, p-chlorobenzyl,m-chlorobenzyl, p-nitrobenzyl, benzyloxybenzyl, pentafluorobenzyl andthe like; amino or alkylamino containing 1 to 20 carbons such as amonoalkyl- or monoaralkylamino groups like methylamino, ethylamino,propylamino or benzylamino and the like, dialkylamino such asdimethylamino, diethylamino, dibenzylamino, pyrrolidino, piperidino ormolpholino and the like.

Thus, in certain embodiments, R¹ is hydrogen, hydroxyl, sulfhydryl,amino, substituted amino, hydroxymethyl, monomethoxy, halogen,pseudohalogen, or a lower hydrocarbon (which hydrocarbon can besubstituted or unsubstituted) containing from 1 to 20 atoms. In aparticular embodiment, R¹ is a lower hydrocarbon selected from alkyl,substituted alkyl, alkenyl, alkanoyl, aryl, aroyl, aralkyl, oralkylamino. In a particular embodiment, R¹ is a lower hydrocarbonsubstituted with alkoxy, substituted alkoxy, imidate, arylthio, or(substituted aryl) thio. In other embodiments, R¹ is a lower alkylselected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl,tert-butyl and pentyl. In other embodiments, R¹ is a lower alkenylselected from vinyl, substituted vinyl, ethynyl, or substituted ethynyl.In other embodiments, R¹ is a lower alkanoyl selected from formyl,acetyl, propionyl, isopropionyl, butyryl, isobutyryl, tert-butyryl,valeryl, pivaloyl, caproyl, capryl, lauryl, myristyl, palmityl, stearyl,arachidyl, stilligyl, palmitoyl, oleyl, linolenyl, and arachidonoyl. Inother embodiments, R¹ is lower aryl selected from phenyl, p-tolyl,p-chlorophenyl, p-aminophenyl, p-nitrophenyl, p-anisyl. In yet otherembodiments, R¹ is a lower aroyl selected from benzoyl and naphthoyl. Inother embodiments, R¹ is a lower aralkyl selected from benzyl,benzhydryl, p-chlorobenzyl, m-chlorobenzyl, p-nitrobenzyl,benzyloxybenzyl, or pentafluorobenzyl. In certain other embodiments, R¹is a lower alkylamino is selected from monoalkylamino, monoaralkylamino,dialkylamino, diaralkylamino, and benzylamino.

Compounds of interest include, but are not limited to, those of formula(I) where R¹ is selected from hydrogen, fluorine, trifluoromethyl,methyl, ethyl, propyl, butyl, isopropyl, isobutyl, acetyl, propionyl,butyryl, 2-bromovinyl, phenyl, benzyl, benzoyl, benzyloxybenzyl,benzylamino, alkyloxyalkyl, benzyloxyalkyl, imidatealkyl, arylthio, and(substituted aryl) thio. Thus, in certain embodiments, the compound isof formula (I), and R¹ is H, F, CF₃, CH₃, CH₃CH₂, CH₃CH₂CH₂, (CH₃)₂CH,(CH₃)₂CH₂CH₂, CH₃(O)CCH₂, CH₃(O)CCH₂CH₂, Br—CH═CH, phenyl, benzyl,benzoyl, benzyloxybenzyl, benzyl-NH—, CH₃CH₂OCH₂, benzyl-O—CH₂, CH₃OCH₂,CH₃C(NH)—O—CH₂, or CH₃-phenyl-O—CH₂.

Examples of R² constituents of interest include, but are not limited to:hydrogen; hydroxyl; sulfhydryl; halogen such as fluorine, chlorine,bromine or iodine, as well as pseudohalogen such as a loweralkylsulfonyl group of 1 to 5 carbons such as methyl-, ethyl-, propyl-,isopropyl-, butyl-, isobutyl-, tert-butyl-, and pentasulfonyl orarylsulfonyl such as benzene, p-toluene, p-nitrobenzenesulfonyl groups;lower alkyl containing 1 to 20 carbons such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl and the like, includingsubstituted lower alkyl such as aminomethyl, hydroxymethyl, methoxy,ethyloxy, propyloxy, and the like; lower alkenyl containing 1 to 20carbons such as vinyl and substituted vinyl, ethynyl and substitutedethynyl, where the substituted vinyl or substituted ethynyl designatessubstitution of the β position of vinyl or ethynyl by a halogen such asbromine, chlorine, fluorine or iodine, or substitution by an alkyl of 1to 5 carbon atoms such as methyl, ethyl, propyl, butyl, pentyl and thelike, or aralkyl such as benzyl, p-chlorobenzyl, p-nitrobenzyl and thelike, or aryl such as phenyl, p-nitrophenyl, p-tolyl, p-anisyl, naphtyland the like; lower alkanoyl (acyl groups) and esters thereof of a mainchain containing 1 to 20 carbons such as formyl, acetyl, propionyl,isopropionyl, butyryl, isobutyryl, tert-butyryl, valeryl, pivaloyl,caproyl, capryl, lauryl, myristyl, palmityl, stearyl, arachidyl,stilligyl, palmitoyl, oleyl, linolenyl, arachidonoyl and the like; loweraryl containing 1 to 20 carbons such as phenyl, p-tolyl, p-chlorophenyl,p-aminophenyl, p-nitrophenyl, p-anisyl and the like; lower aroylcontaining 1 to 20 carbons such as benzoyl and naphthoyl, where thearomatic group may be additionally substituted by alkyl, alkoxy, halo,or nitro moieties such as p-tolnoyl, p-anisoyl, p-chlorobenzoyl,p-nitrobenzoyl or 2,4-dinitrobenzoyl, pentafluorobenzoyl and the like,or another aroyl such as benzyloxybenzyl and the like; lower aralkylcontaining 1 to 20 carbons such as benzyl, benzhydryl, p-chlorobenzyl,m-chlorobenzyl, p-nitrobenzyl, benzyloxybenzyl, pentafluorobenzyl andthe like; lower aryloxy containing 1 to 20 carbons such as phenyloxy(ie, O-phenyl), benzyloxy (ie, O-benzyl), benzhydryloxy (ie,O-benzhydryl), p-chlorobenzyloxy (ie, O-(p-chlorobenzyl)),m-chlorobenzyloxy (ie, O-(m-chlorobenzyl)), p-nitrobenzyloxy (ie,O-(p-nitrobenzyl)), (4-benzyloxybenzyl)-oxy (ie, O-benzyloxybenzyl), orpentafluorobenzyloxy (ie, O-pentafluorobenzyl); esters of aryloxys, suchas lower aryloxy (ie, O-aroyl) containing 1 to 20 carbons such asbenzoyloxy (ie, O-benzoyl), diphenylacetyloxy (ie, O-diphenylacetyl),p-chlorobenzoyloxy (ie, O-(p-chlorobenzoyl)), m-chlorobenzoyloxy (ie,O-(m-chlorobenzoyl)), p-nitrobenzoyloxy (ie, O-(p-nitrobenzoyl)),(4-benzyloxybenzyl)-oxy (ie, O-benzyloxybenzyl), or pentafluorobenzyloxy(ie, O-pentafluorobenzoyl); amino or alkylamino containing 1 to 20carbons such as a monoalkyl- or monoaralkylamino groups likemethylamino, ethylamino, propylamino or benzylamino and the like,dialkylamino such as dimethylamino, diethylamino, dibenzylamino,pyrrolidino, piperidino or molpholino and the like.

Thus, in certain embodiments, R² is hydrogen, hydroxyl, sulfhydryl,amino, hydroxymethyl, monomethoxy, halogen, pseudohalogen, or a lowerhydrocarbon (which hydrocarbon can be substituted or unsubstituted)containing from 1 to 20 atoms, and esters thereof. In a particularembodiment, R² is a lower hydrocarbon selected from alkyl, alkenyl,alkanoyl, aryl, aroyl, aryloxy, aryloxy, aralkyl, or alkylamino. Inother embodiments, R² is a lower alkyl selected from methyl, ethyl,propyl, isopropyl, butyl, isobutyl, tert-butyl and pentyl. In otherembodiments, R² is a lower alkenyl selected from vinyl, substitutedvinyl, ethynyl, or substituted ethynyl. In other embodiments, R² is alower alkanoyl selected from formyl, acetyl, propionyl, isopropionyl,butyryl, isobutyryl, tert-butyryl, valeryl, pivaloyl, caproyl, capryl,lauryl, myristyl, palmityl, stearyl, arachidyl, stilligyl, palmitoyl,oleyl, linolenyl, and arachidonoyl. In other embodiments, R² is loweraryl selected from phenyl, p-tolyl, p-chlorophenyl, p-aminophenyl,p-nitrophenyl, p-anisyl. In yet other embodiments, R² is a lower aroylselected from benzoyl and naphthoyl. In other embodiments, R² is a loweraralkyl selected from benzyl, benzhydryl, p-chlorobenzyl,m-chlorobenzyl, p-nitrobenzyl, benzyloxybenzyl, or pentafluorobenzyl. Inother embodiments, R² is a lower aryloxy selected from phenyloxy,benzyloxy, benzhydryloxy, p-chlorobenzyloxy, m-chlorobenzyloxy,p-nitrobenzyloxy, (4-benzyloxybenzyl)-oxy, or pentafluorobenzyloxy. Inother embodiments, R² is a lower aroyloxy selected from benzoyloxy,diphenylacetyloxy, p-chlorobenzoyloxy, m-chlorobenzoyloxy,p-nitrobenzoyloxy, (4-benzyloxybenzyl)-oxy, or pentafluorobenzyloxy. Incertain other embodiments, R² is a lower alkylamino is selected frommonoalkylamino, monoaralkylamino, dialkylamino, and diaralkylamino.Thus, in certain embodiments, R² can not only be hydrogen or hydroxyl,but also an O-acyl, alkoxy, alkoxycarbonyl, alkoxycarbonylamino,O-alkyl, O-alkylene, O-alkynyl, O-aralkyl, O-aryl, O-aryloxy,O-carbohydrate, O-cycloalkenyl, O-cycloalkyl, O-heterocycloalkyl,O-heteroaryl. In addition, an S can substitute for the O.

Compounds of interest include, but are not limited to, those of formula(I) where R² is selected from hydrogen, fluorine, trifluoromethyl,methyl, ethyl, propyl, butyl, isopropyl, isobutyl, acetyl, propionyl,butyryl, 2-bromovinyl, phenyl, phenyloxy, benzyl, benzoyl, benzoyloxyand benzyloxybenzyl. Thus, in certain embodiments, the compound is offormula (I), and R² is H, F, CF₃, CH₃, CH₃CH₂, CH₃CH₂CH₂, (CH₃)₂CH,(CH₃)₂CH₂CH₂, CH₃(O)CCH₂, CH₃(O)CCH₂CH₂, Br—CH═CH, phenyl, phenyloxy,benzyl, benzoyl, benzoyloxy, or benzyloxybenzyl.

In specific embodiments of interest, the compound is of formula (I), andR² is hydrogen, hydroxyl, or an O-linked substituent. This includescompounds of formula (I), where R² is H, OH or C₆H₅C(O)O.

Examples of R³ of interest include, but are not limited to: hydrogen;hydroxyl; azido; sulfhydryl; halogen; pseudohalogen; lower alkylcontaining 1 to 20 carbons such as methyl, ethyl, propyl, isopropyl,butyl, isobutyl, tert-butyl, pentyl and the like, including asubstituted lower alkyl such as aminomethyl, hydroxymethyl, methoxy,ethyloxy, propyloxy, and the like; lower alkanoyl (acyl) includingesters thereof of a main chain of 1 to 20 carbon atoms such as formyl,acetyl, propionyl, isopropionyl, butyryl, isobutyryl, tert-butyryl,valeryl, pivaloyl, caproyl, capryl, lauryl, myristyl, palmityl, stearyl,arachidyl, stilligyl, palmitoyl, oleyl, linolenyl, arachidonoyl and thelike; lower aryl such as phenyl, p-nitrophenyl, p-tolyl, p-anisyl,naphtyl and the like; lower aroyl (acyl radical of an aromatic acid) of1 to 20 carbons such as benzoyl and naphthoyl, where the aromatic groupmay be additionally substituted by alkyl, alkoxy, halo, or nitromoieties such as p-tolnoyl, p-anisoyl, p-chlorobenzoyl, p-nitrobenzoylor 2,4-dinitrobenzoyl, pentafluorobenzoyl and the like; lower aryloxy of1 to 20 carbons such as phenyloxy, benzyloxy, benzhydryloxy,p-chlorobenzyloxy, m-chlorobenzyloxy, p-nitrobenzyloxy,(4-benzyloxybenzyl)-oxy, or pentafluorobenzyloxy and the like; as wellas esters of aryloxys, such as lower aroyloxy (O-aroyls) of 1 to 20carbons such as benzoyloxy, diphenylacetyloxy, p-chlorobenzoyloxy,m-chlorobenzoyloxy, p-nitrobenzoyloxy, (4-benzyloxybenzyl)-oxy, orpentafluorobenzyloxy and the like. R³ may also be adamantyl, orsubstituted adamantyl.

Thus, in certain embodiments, R³ is hydrogen, hydroxyl, azido,sulfhydryl, hydroxymethyl, halogen, or pseudohalogen. In otherembodiments, R³ is a lower hydrocarbon selected from alkyl, alkanoyl,aryl, aroyl, aryloxy, aroyloxy, or aralkyl. In other embodiments, R³ isa lower alkyl selected from methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl and pentyl. In other embodiments, R³ is a loweralkanoyl selected from formyl, acetyl, propionyl, isopropionyl, butyryl,isobutyryl, tert-butyryl, valeryl, pivaloyl, caproyl, capryl, lauryl,myristyl, palmityl, stearyl, arachidyl, stilligyl, palmitoyl, oleyl,linolenyl, and arachidonoyl. In other embodiments, R³ is a lower arylselected from phenyl, p-tolyl, p-chlorophenyl, p-aminophenyl,p-nitrophenyl, p-anisyl and the like. In other embodiments, R³ is alower aroyl selected from benzoyl and naphthoyl. In yet other certainembodiments, R³ is a lower aralkyl selected from benzyl, benzhydryl,p-chlorobenzyl, m-chlorobenzyl, p-nitrobenzyl, benzyloxybenzyl, orpentafluorobenzyl. In other embodiments, R³ is a lower aryloxy selectedfrom phenyloxy, benzyloxy, benzhydryloxy, p-chlorobenzyloxy,m-chlorobenzyloxy, p-nitrobenzyloxy, (4-benzyloxybenzyl)-oxy, orpentafluorobenzyloxy. In other embodiments, R³ is a lower aroyloxyselected from benzoyloxy, diphenylacetyloxy, p-chlorobenzoyloxy,m-chlorobenzoyloxy, p-nitrobenzoyloxy, (4-benzyloxybenzyl)-oxy, orpentafluorobenzyloxy. Thus, in certain embodiments, R³ can not only behydrogen or hydroxyl, but also an O-acyl, alkoxy, alkoxycarbonyl,alkoxycarbonylamino, O-alkyl, O-alkylene, O-alkynyl, O-aralkyl, O-aryl,O-aryloxy, O-carbohydrate, O-cycloalkenyl, O-cycloalkyl,O-heterocycloalkyl, O-heteroaryl. In addition, an S can substitute forthe O.

Compounds of interest are those of formula (I) where R³ is hydrogen,hydroxyl, halogen, azido, or an O-linked substituent. This includescompounds of formula (I) where R³ is selected from hydrogen, hydroxyl,n-butoxy, isobutyloxy, t-butyloxy, phenyloxy, benzyloxy, benzoyloxy, andpentafluorobenzyloxy. Thus, in certain embodiments, the compound is offormula (I), and R³ is selected from H, OH, CH₃CH₂CH₂CH₂O,(CH₃)₂CH₂CH₂O, (CH₃)₃CO, C₆H₅O, benzoyloxy, and pentafluorobenzyloxy.

In specific embodiments of interest, the compound is of formula (I),where R³ is H, OH, F, Cl, Br, I, N₃, or C₆H₅C(O)O. Of special interestis a compound of formula (I), where R³ is OH, or O-acyl (for example, anester such as C₆H₅C(O)O).

Examples of R⁴ include, but are not limited to: hydrogen; hydroxyl;sulfhydryl; halogen such as fluorine, chlorine, bromine or iodine; aminoor lower alkylamino. R⁴ also is exemplified by lower alkyl, with acylgroups which may be lower alkanoyl groups of 1 to 7 carbon atoms such asformyl, acetyl, propionyl, isopropionyl, butyryl, isobutyryl,tert-butyryl and the like, and esters thereof. Thus, R⁴ can also bearoyl (and esters thereof such as O-linked aroyls, ie, O-arolys orarolyoxy) such as benzoyl and naphthoyl wherein the aromatic group maybe additionally substituted by alkyl, alkoxy, halo, or nitro moietiessuch as p-tolnoyl, p-anisoyl, p-chlorobenzoyl, p-nitrobenzoyl or2,4-dinitrobenzoyl and the like. Accordingly, in certain embodiments, R⁴can not only be hydrogen or hydroxyl, but also an O-acyl, alkoxy,alkoxycarbonyl, alkoxycarbonylamino, O-alkyl, O-alkylene, O-alkynyl,O-aralkyl, O-aryl, O-aryloxy, O-carbohydrate, O-cycloalkenyl,O-cycloalkyl, O-heterocycloalkyl, O-heteroaryl. In addition, an S cansubstitute for the O.

Thus, in certain embodiments, R⁴ is hydrogen; hydroxyl; sulfhydryl;halogen, amino aminomethyl, or aminodimethyl. In other embodiments, R⁴is a lower alkyl, acyl, aroyl, or aroyloxy. This includes a specificembodiment, where the compound of formula (I) is one where R⁴ ishydrogen, flourine, hydroxyl, amino, aminomethyl, aminodimethyl,t-butyloxy, phenyloxy or benzoyloxy (for example, a compound of formula(I), where R⁴ is H, F, OH, NH₂, NHCH₃, N(CH₃)₂, (CH₃)₃CO, C₆H₅O orC₆H₅C(O)O).

Compounds of particular interest are those of formula (I) where R⁴ ishydrogen, hydroxyl, or an O-linked substituent. In specific embodiments,the compound is of formula (I), where R⁴ is H, OH or C₆H₅C(O)O. Ofspecial interest is a compound of formula (I), where R⁴ is OH, or O-acyl(for example, an ester such as C₆H₅C(O)O).

Of interest are compounds of formula (I) where: R¹ is H, F, CF₃, CH₃,CH₃CH₂, CH₃CH₂CH₂, (CH₃)₂CH, (CH₃)₂CH₂CH₂, CH₃(O)CCH₂, CH₃(O)CCH₂CH₂,Br—CH═CH, phenyl, benzyl, benzoyl, or benzyloxybenzyl, R² is H, OH, F,CF₃, CH₃, CH₃CH₂, CH₃CH₂CH₂, (CH₃)₂CH, (CH₃)₂CH₂CH₂, CH₃(O)CCH₂,CH₃(O)CCH₂CH₂, Br—CH═CH, phenyl, phenyloxy, benzyl, benzoyl, benzoyloxy,or benzyloxybenzyl, and where R³ and R⁴ are each hydroxyl. These includethe compounds: 2,2′-anhydrouridine; 2,2′-anhydro-5-fluorouridine;2,2′-anhydro-5-trifluoromethyluridine; 2,2′-anhydro-5-methyluridine;2,2′-anhydro-5-ethyluridine; 2,2′-anhydro-5-propyluridine;2,2′-anhydro-5-isopropyluridine; 2,2′-anhydro-5-isobutyluridine;2,2′-anhydro-5-methylacyluridine; 2,2′-anhydro-5-propylacyluridine;2,2′-anhydro-5-(2-bromovinyl)-uridine; 2,2′-anhydro-5-phenylluridine;2,2′-anhydro-5-benzoyluridine; 2,2′-anhydro-5-benzoyluridine; and2,2′-anhydro-5-(benzyloxybenzyl)-uridine. Of special interest is2,2′-anhydro-5-methyluridine, or the pharmaceutically acceptable salts,solvates, hydrates, and prodrug forms thereof, and stereoisomersthereof.

Additional compounds of interest are compounds of formula (I) where: R¹is H, F, CF₃, CH₃, CH₃CH₂, CH₃CH₂CH₂, (CH₃)₂CH, (CH₃)₂CH₂CH₂,CH₃(O)CCH₂, CH₃(O)CCH₂CH₂, Br—CH═CH, phenyl, benzyl, benzoyl, orbenzyloxybenzyl, R² is H, OH, F, CF₃, CH₃, CH₃CH₂, CH₃CH₂CH₂, (CH₃)₂CH,(CH₃)₂CH₂CH₂, CH₃(O)CCH₂, CH₃(O)CCH₂CH₂, Br—CH═CH, phenyl, phenyloxy,benzyl, benzyloxy, benzoyl, benzoyloxy, or benzyloxybenzyl, and where R³is hydroxyl, and R⁴ is benzoyloxy. These include the compounds:3′-O-benzoyl-2,2′-anhydrouridine;3′-O-benzoyl-2,2′-anhydro-5-fluorouridine;3′-O-benzoyl-2,2′-anhydro-5-trifluoromethyluridine;3′-O-benzoyl-2,2′-anhydro-5-methyluridine;3′-O-benzoyl-2,2′-anhydro-5-ethyluridine;3′-O-benzoyl-2,2′-anhydro-5-propyluridine;3′-O-benzoyl-2,2′-anhydro-5-isopropyluridine;3′-O-benzoyl-2,2′-O-anhydro-5-isobutyluridine;3′-O-benzoyl-2,2′-anhydro-5-methylacyluridine;3′-O-benzoyl-2,2′-anhydro-5-propylacyluridine;3′-O-benzoyl-2,2′-anhydro-5-(2-bromovinyl)-uridine;3′-O-benzoyl-2,2′-anhydro-5-phenylluridine;3′-O-benzoyl-2,2′-anhydro-5-benzoyluridine;3′-O-benzoyl-2,2′-anhydro-5-benzoyluridine; and3′-O-benzoyl-2,2′-anhydro-5-(benzyloxybenzyl)-uridine. Of specificinterest is 3′-O-benzoyl-2,2′-anhydro-5-methyluridine, or thepharmaceutically acceptable salts, solvates, hydrates, and prodrug formsthereof, and stereoisomers thereof.

Also of interest are compounds of formula (I) where: R¹ is H, F, CF₃,CH₃, CH₃CH₂, CH₃CH₂CH₂, (CH₃)₂CH, (CH₃)₂CH₂CH₂, CH₃(O)CCH₂,CH₃(O)CCH₂CH₂, Br—CH═CH, phenyl, benzyl, benzoyl, or benzyloxybenzyl, R²is H, OH, F, CF₃, CH₃, CH₃CH₂, CH₃CH₂CH₂, (CH₃)₂CH, (CH₃)₂CH₂CH₂,CH₃(O)CCH₂, CH₃(O)CCH₂CH₂, Br—CH═CH, phenyl, phenyloxy, benzyl,benzyloxy, benzoyl, benzoyloxy, or benzyloxybenzyl, and where R³ isbenzoyloxy, and R⁴ is hydroxyl. These include the compounds:5′-O-benzoyl-2,2′-anhydrouridine;5′-O-benzoyl-2,2′-anhydro-5-fluorouridine;5′-O-benzoyl-2,2′-anhydro-5-trifluoromethyluridine;5′-O-benzoyl-2,2′-anhydro-5-methyluridine;5′-O-benzoyl-2,2′-anhydro-5-ethyluridine;5′-O-benzoyl-2,2′-anhydro-5-propyluridine;5′-O-benzoyl-2,2′-anhydro-5-isopropyluridine;5′-O-benzoyl-2,2′-O-anhydro-5-isobutyluridine;5′-O-benzoyl-2,2′-anhydro-5-methylacyluridine;5′-O-benzoyl-2,2′-anhydro-5-propylacyluridine;5′-O-benzoyl-2,2′-anhydro-5-(2-bromovinyl)-uridine;5′-O-benzoyl-2,2′-anhydro-5-phenylluridine;5′-O-benzoyl-2,2′-anhydro-5-benzoyluridine;5′-O-benzoyl-2,2′-anhydro-5-benzoyluridine; and5′-O-benzoyl-2,2′-anhydro-5-(benzyloxybenzyl)-uridine. Of specificinterest is 5′-O-benzoyl-2,2′-anhydro-5-methyluridine, or thepharmaceutically acceptable salts, solvates, hydrates, and prodrug formsthereof, and stereoisomers thereof.

The 2,2′-anhydropyrimidine compounds of the invention may be incompositions that contain single stereoisomers, mixtures ofstereoisomers, as well various derivatives thereof that can occur asequilibrium mixtures of tautomers. For instance, 2,2′-anhydropyrimidinesaccording to formula (I) include four stereo centers with respect to thefurano ring, which includes the α and β anomers, and the L or D mirrorimage configurations. Examples of stereoisomers of the2,2′-anhydropyrimidine compounds of the invention are the β-D-isomer,β-L-isomer, α-D-isomer, and α-L-isomer, as well as tautomers andmixtures including α,β-D-isomers, α,β-L-isomers, α-DL-isomers, andβ-DL-isomers. Thus, in one embodiment, compositions are provided thatconsists essentially of a stereoisomer of a 2,2′-anhydropyrimidine thatis a β-D-isomer, β-L-isomer, α-D-isomer, or an α-L-isomer.

Stereoisomers of particular interest include:2,2′-anhydro-1-(ρ-D-arabinofuranosyl)uracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-fluorouracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-trifluoromethyluracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyluracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-ethyluracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-n-propyluracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-isopropyluracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-isobutyluracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyacyluracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-propylacyluracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-(2-bromovinyl)uracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-phenyluracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-benzyluracil;2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-benzoyluracil; and2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-(3-benzyloxybenzyl)uracil.Further stereoisomers of interest include:3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)uracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-fluoruracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-trifluoromethyluracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyluracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-ethyluracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-n-propyluracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-isopropyluracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-isobutyluracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyacyluracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-propylacyluracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-(2-bromovinyl)uracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-phenyluracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-benzyluracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-benzoyluracil; and3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-(3-benzyloxybenzyl)uracil.Additional stereoisomers of interest include:5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)uracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-fluorouracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-trifluoromethyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-ethyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-n-propyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-isopropyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-isobutyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyacyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-propylacyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-(2-bromovinyl)uracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-phenyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-benzyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-benzoyluracil; and5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-(3-benzyloxybenzyl)uracil.

Examples of other analogs or derivatives of the 2,2′-anhydropyrimidinesof the invention, and stereoisomers thereof include:3′-O-acetyl-2,2′-anhydro-5-propyluridine(3′-O-acetyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-propyluracil); and3′-O-acetyl-2,2′-anhydro-5-isopropyluridine(3′-O-acetyl-2,2′-anhydro-1-(1-D-arabinofuranosyl)-5-isopropyluracil);as well as the 2,2′-anhydrocytidines, and analogs and derivativesthereof, of which the stereoisomer2,2′-anhydro-1-(β-D-arabinofuranosyl)cytosine is one example.

As noted above, stereoisomers and the various 2,2′-anhydropyrimidines ofparticular interest are those which exhibit improved activity on a molarbasis. Such compounds can be readily selected for this purpose bycomparing against a matrix of compounds of particular interest, such asthose illustrated in Table 4 (where the compound is of formula (I)).

TABLE 4 The compound is of formula (I) Compound Stereoisomer R¹ R² R³ R⁴I-a β-D-isomer H H OH OH I-b β-D-isomer CH₃ H OH OH I-c β-D-isomerCH₃CH₂ H OH OH I-d β-D-isomer CH₃CH₂CH H OH OH I-e β-D-isomer BrCH═CH HOH OH I-f β-D-isomer C₆H₅CH₂ H OH OH I-g β-D-isomer H H C₆H₅C(O)O OH I-hβ-D-isomer CH₃ H C₆H₅C(O)O OH I-i β-D-isomer CH₃CH₂ H C₆H₅C(O)O OH I-jβ-D-isomer CH₃CH₂CH H C₆H₅C(O)O OH I-k β-D-isomer BrCH═CH H C₆H₅C(O)O OHI-l β-D-isomer C₆H₅CH₂ H C₆H₅C(O)O OH I-m β-D-isomer F—C₆H₅CH₂ H OH OHI-n β-D-isomer NO₂—C₆H₅CH₂ H OH OH I-o β-D-isomer NH₂—C₆H₅CH₂ H OH OHI-p β-D-isomer Cl—C₆H₅CH₂ H OH OH I-q β-D-isomer Alkyl-C₆H₅CH₂ H OH OHI-r β-D-isomer Methoxy-C₆H₅CH₂ H OH OH I-s β-D-isomer Thiol-C₆H₅CH₂ H OHOH I-t β-D-isomer F—C₆H₅CH₂ H C₆H₅C(O)O OH I-u β-D-isomer NO₂—C₆H₅CH₂ HC₆H₅C(O)O OH I-v β-D-isomer NH₂—C₆H₅CH₂ H C₆H₅C(O)O OH I-w β-D-isomerCl—C₆H₅CH₂ H C₆H₅C(O)O OH I-x β-D-isomer Alkyl-C₆H₅CH₂ H C₆H₅C(O)O OHI-y β-D-isomer Methoxy- H C₆H₅C(O)O OH C₆H₅CH₂ I-z β-D-isomerThiol-C₆H₅CH₂ H C₆H₅C(O)O OH I-a′ β-D-isomer H OH H OH I-b′ β-D-isomerCH₃ OH H OH I-c′ β-D-isomer CH₃CH₂ OH H OH I-d′ β-D-isomer CH₃CH₂CH OH HOH I-e′ β-D-isomer BrCH═CH OH H OH I-f′ β-D-isomer C₆H₅CH₂ OH H OH I-g′β-D-isomer H C₆H₅C(O)O H OH I-h′ β-D-isomer CH₃ C₆H₅C(O)O H OH I-I′β-D-isomer CH₃CH₂ C₆H₅C(O)O H OH I-j′ β-D-isomer CH₃CH₂CH C₆H₅C(O)O H OHI-k′ β-D-isomer BrCH═CH C₆H₅C(O)O H OH I-l′ β-D-isomer C₆H₅CH₂ C₆H₅C(O)OH OH I-m′ β-D-isomer F—C₆H₅CH₂ OH H OH I-n′ β-D-isomer NO₂—C₆H₅CH₂ OH HOH I-o′ β-D-isomer NH₂—C₆H₅CH₂ OH H OH I-p′ β-D-isomer Cl—C₆H₅CH₂ OH HOH I-q′ β-D-isomer Alkyl-C₆H₅CH₂ OH H OH I-r′ β-D-isomer Methoxy- OH HOH C₆H₅CH₂ I-s′ β-D-isomer Thiol-C₆H₅CH₂ OH H OH I-t′ β-D-isomerF—C₆H₅CH₂ C₆H₅C(O)O H OH I-u′ β-D-isomer NO₂—C₆H₅CH₂ C₆H₅C(O)O H OH I-v′β-D-isomer NH₂—C₆H₅CH₂ C₆H₅C(O)O H OH I-w′ β-D-isomer Cl—C₆H₅CH₂C₆H₅C(O)O H OH I-x′ β-D-isomer Alkyl-C₆H₅CH₂ C₆H₅C(O)O H OH I-y′β-D-isomer Methoxy- C₆H₅C(O)O H OH C₆H₅CH₂ I-z′ β-D-isomer Thiol-C₆H₅CH₂C₆H₅C(O)O H OH

As mentioned above, the compounds in Table 4 are illustrative but notlimiting. For example, R⁴ can be not only hydroxyl, but also an O-acyl,alkoxy, alkoxycarbonyl, alkoxycarbonylamino, O-alkyl, O-alkylene,O-alkynyl, O-aralkyl, O-aryl, O-aryloxy, O-carbohydrate, O-cycloalkenyl,O-cycloalkyl, O-heterocycloalkyl, O-heteroaryl. In addition, an S cansubstitute for the O and other combinations of the structural elementssuch as described herein, as well as other stereochemical orientations,are also possible.

In certain embodiments, acyl derivatives of the 2,2′-anyhydropyrimidinesof formula (I) are of interest. Thus, compounds of formula (I) includethose in which R¹, R², R³ and R⁴ are as defined above, wherein at leastone of R², R³ and R⁴ is an acyl derivative. By “acyl derivative” isintended a derivative of a 2,2′-anyhydropyrimidine of formula (I) inwhich at least one of R², R³ and R⁴ is a substantially nontoxic organicacyl substituent obtainable from a carboxylic acid that is attached to ahydroxyl group on the ribose or pyrimidine ring of formula (I) throughan ester linkage.

Acyl derivatives of a 2,2′-anyhydropyrimidine compound of formula (I)include those in which R¹ is as defined above, and each R², R³ and R⁴ isindependently hydrogen, hydroxyl or an acyl radical, with the provisothat at least one of R², R³ and R⁴ is not hydrogen. In anotherembodiment, the acyl derivative of a 2,2′-anyhydropyrimidine is acompound of formula (I) in which R¹ and R² are as defined above, withthe proviso that R² is other than hydrogen, and each R³ and R⁴ isindependently hydroxyl or an acyl radical. In one embodiment, the acylderivative of a 2,2′-anyhydropyrimidine is a compound of formula (I) inwhich R¹ is as defined above, R² is hydrogen, and each R³ and R⁴ isindependently hydroxyl or an acyl radical. Of particular interest, is anacyl derivative of a 2,2′-anyhydropyrimidine compound of formula (I),wherein R¹ is methyl, R² is hydrogen, and each R³ and R⁴ isindependently hydroxyl or an acyl radical. Also of interest is an acylderivative of a 2,2′-anyhydropyrimidine compound of formula (I), whereinR¹ is methyl, R² is hydrogen, and each R³ and R⁴ is an acyl radical.

In general, the ester linkage(s) of an acyl derivative of formula (I)are cleavable under physiological conditions, either in vitro, such asin a cell-based system, and/or in vivo, such as through metabolism in abody. Thus, in certain embodiments, the acyl radical is a radical of ametabolite. Such acyl substituents include, but are not limited to,those derived from acetic acid, fatty acids, amino acids, lipoic acid,glycolic acid, lactic acid, enolpyruvic acid, pyruvic acid, orotic acid,acetoacetic acid, beta-hydroxybutyric acid, creatinic acid, succinicacid, fumaric acid, adipic acid, benzoic acid and p-aminobenzoic acid.Particular acyl substituents of interest are compounds which arenormally present in the body, either as dietary constituents or asintermediary metabolites, and which are essentially nontoxic whencleaved from the 2,2′-anyhydropyrimidine compound of interest in vivo.

Of particular interest are compositions comprising a3′-O-acyl-2,2′-anhydropyrimidine or derivative thereof. For example,acyl derivatives of interest are those that include a2,2′-anyhydropyrimidine compound of formula (I), where each R¹, R² andR³ is independently selected from selected from hydrogen, hydroxyl,sulfhydryl, amino, hydroxymethyl, methoxy, halogen, pseudohalogen, and asubstituted or unsubstituted lower hydrocarbon containing 1 to 20carbons, such as a lower hydrocarbon selected from alkyl, alkenyl,alkanoyl, aryl, aroyl, aralkyl and alkylamino, and esters thereof, andwhere R⁴ is an O-acyl radical.

In certain embodiments, the acyl derivatives include a2,2′-anyhydropyrimidine compound of formula (I), where R⁴ is an O-acylradical, and where the O-acyl radical comprises 1 to 10 carbon atoms,such as an O-acyl radical selected from aroyloxy, aralkyloxy,heteroaryoloxy, and cycloalkyloxy.

Accordingly, acyl derivatives of a 2,2′-anyhydropyrimidine compound offormula (I) include 3′-O-acyl-2,2′-anyhdropyrimidines,5′-O-acyl-2,2′-anyhdropyrimidines, 3′,5′-O-acyl-2,2′-anyhdropyrimidines,and derivatives thereof. For example, 3′-O-acyl-2,2′-anhydropyrimidinesor derivatives thereof include 3′-O-aroyl-2,2′-anhydropyrimidines, suchas a 3′-O-aroyl-2,2′-anhydrouridine or derivative thereof. An example ofparticular interest is 3′-O-benzoyl-2,2′-anhydrouridine or derivativethereof, such as 3′-O-benzoyl-2,2′-anhydro-5-methyluridine. Also ofinterest is a compound in which the3′-O-benzoyl-2,2′-anhydro-5-methyluridine is the stereoisomer3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyluracil.

In some embodiments, acyl derivatives of a 2,2′-anyhydropyrimidinecompound of formula (I) include those where: R¹ is H, F, CF₃, CH₃,CH₃CH₂, CH₃CH₂CH₂, (CH₃)₂CH, (CH₃)₂CH₂CH₂, CH₃(O)CCH₂, CH₃(O)CCH₂CH₂,Br—CH═CH, phenyl, benzyl, benzoyl, or benzyloxybenzyl, R² is H, OH, F,CF₃, CH₃, CH₃CH₂, CH₃CH₂CH₂, (CH₃)₂CH, (CH₃)₂CH₂CH₂, CH₃(O)CCH₂,CH₃(O)CCH₂CH₂, Br—CH═CH, phenyl, phenyloxy, benzyl, benzyloxy, benzoyl,benzyloxybenzyl, or acyl radical, and where each R³ and R⁴ isindependently hydroxyl or an acyl radical. These include the compounds:3′-O-benzoyl-2,2′-anhydrouridine;3′-O-benzoyl-2,2′-anhydro-5-fluorouridine;3′-O-benzoyl-2,2′-anhydro-5-trifluoromethyluridine;3′-O-benzoyl-2,2′-anhydro-5-methyluridine;3′-O-benzoyl-2,2′-anhydro-5-ethyluridine;3′-O-benzoyl-2,2′-anhydro-5-propyluridine;3′-O-benzoyl-2,2′-anhydro-5-isopropyluridine;3′-O-benzoyl-2,2′-O-anhydro-5-isobutyluridine;3′-O-benzoyl-2,2′-anhydro-5-methylacyluridine;3′-O-benzoyl-2,2′-anhydro-5-propylacyluridine;3′-O-benzoyl-2,2′-anhydro-5-(2-bromovinyl)-uridine;3′-O-benzoyl-2,2′-anhydro-5-phenylluridine;3′-O-benzoyl-2,2′-anhydro-5-benzoyluridine;3′-O-benzoyl-2,2′-anhydro-5-benzoyluridine; and3′-O-benzoyl-2,2′-anhydro-5-(benzyloxybenzyl)-uridine;5′-O-benzoyl-2,2′-anhydrouridine;5′-O-benzoyl-2,2′-anhydro-5-fluorouridine;5′-O-benzoyl-2,2′-anhydro-5-trifluoromethyluridine;5′-O-benzoyl-2,2′-anhydro-5-methyluridine;5′-O-benzoyl-2,2′-anhydro-5-ethyluridine;5′-O-benzoyl-2,2′-anhydro-5-propyluridine;5′-O-benzoyl-2,2′-anhydro-5-isopropyluridine;5′-O-benzoyl-2,2′-O-anhydro-5-isobutyluridine;5′-O-benzoyl-2,2′-anhydro-5-methylacyluridine;5′-O-benzoyl-2,2′-anhydro-5-propylacyluridine;5′-O-benzoyl-2,2′-anhydro-5-(2-bromovinyl)-uridine;5′-O-benzoyl-2,2′-anhydro-5-phenylluridine;5′-O-benzoyl-2,2′-anhydro-5-benzoyluridine;5′-O-benzoyl-2,2′-anhydro-5-benzoyluridine; and5′-O-benzoyl-2,2′-anhydro-5-(benzyloxybenzyl)-uridine;3′,5′-O-benzoyl-2,2′-anhydrouridine;3′,5′-O-benzoyl-2,2′-anhydro-5-fluorouridine;3′,5′-O-benzoyl-2,2′-anhydro-5-trifluoromethyluridine;3′,5′-O-benzoyl-2,2′-anhydro-5-methyluridine;3′,5′-O-benzoyl-2,2′-anhydro-5-ethyluridine;3′,5′-O-benzoyl-2,2′-anhydro-5-propyluridine;3′,5′-O-benzoyl-2,2′-anhydro-5-isopropyluridine;3′,5′-O-benzoyl-2,2′-O-anhydro-5-isobutyluridine;3′,5′-O-benzoyl-2,2′-anhydro-5-methylacyluridine;3′,5′-O-benzoyl-2,2′-anhydro-5-propylacyluridine;3′,5′-O-benzoyl-2,2′-anhydro-5-(2-bromovinyl)-uridine;3′,5′-O-benzoyl-2,2′-anhydro-5-phenylluridine;3′,5′-O-benzoyl-2,2′-anhydro-5-benzoyluridine;3′,5′-O-benzoyl-2,2′-anhydro-5-benzoyluridine; and3′,5′-O-benzoyl-2,2′-anhydro-5-(benzyloxybenzyl)-uridine; or thepharmaceutically acceptable salts, solvates, hydrates, and prodrug formsthereof, and stereoisomers thereof.

Of specific interest is 3′-O-benzoyl-2,2′-anhydro-5-methyluridine,5′-O-benzoyl-2,2′-anhydro-5-methyluridine, and3′,5′-O-benzoyl-2,2′-anhydro-5-methyluridine, or the pharmaceuticallyacceptable salts, solvates, hydrates, and prodrug forms thereof, andstereoisomers thereof. Of specific interest are the β-D-arabinofuranosylisomers of these compounds, or the pharmaceutically acceptable salts,solvates, hydrates, and prodrug forms thereof.

In another embodiment, compounds according to formula (I) of specificinterest are those where R¹ and R⁴ are as defined above, and R² and/orR³ is a cyclic hydrocarbyl. By “cyclic hydrocarbyl” is intended ahydrocarbon-based ring structure having from 3 to about 10 carbon atoms,and having a single cyclic ring or multiple condensed rings that may besubstituted. Cyclic hydrocarbyls of interest are selected from aryl,aralkyl, aryloxy, aroyl, aroyloxy, heteroaryl, heteroaryloxy,heteroaryoloxy, cycloalkyl, cycloalkyloxy and cycloalkyloxy. Thus,cyclic hydrocarbyls of special interest are O-linked to the ribose orpyrimidine ring of formula (I). Compounds where R² and/or R³ is a cyclichydrocarbyl exhibit improved activity on a molar basis.

Accordingly, certain compounds of the invention comprise a 5′-O-(cyclichydrocarbyl)-2,2′-anhydropyrimidine or derivative thereof. Thisembodiment includes 5′-O-(cyclic hydrocarbyl)-2,2′-anhydro-5(R⁵)-uridineor derivatives thereof, where R⁵ is R¹ (eg, R⁵═R¹ where “5(R⁵)” refersto, and is the same as, R¹ of formula (I)).

A compound of interest is 5′-O-aryl-2,2′-anhydropyrimidine or derivativethereof, of which various 2,2′-anhydrouridine derivatives are ofincluded. This includes compounds where the5′-O-aryl-2,2′-anhydropyrimidine is a 5′-O-aroyl-2,2′-anhydropyrimidine,such as: 5′-O-benzoyl-2,2′-anhydropyrimidine;5′-O-chlorobenzyl-2,2′-anhydropyrimidine;5′-O-nitrobenzyl-2,2′-anhydropyrimidine;5′-O-hydroxybenzyl-2,2′-anhydropyrimidine, and the like.

In one embodiment, compounds that exhibit improved activity on a molarbasis or improved specificity with respect to not interfering withfluorouracil therapy efficacy are the 5′-O-aryl-2,2′-anhydrouridines,5′-O-aroyl-2,2′-anhydrouridines, and derivatives thereof, such as5′-O-aryl-2,2′-anhydro-5(R⁴)-uridine,5′-O-aroyl-2,2′-anhydro-5(R⁴)-uridine, and their derivatives. Examplesinclude 5′-O-aryl-2,2′-anhydro-5-methyl-uridine;5′-O-aryl-2,2′-anhydro-5-ethyl-uridine;5′-O-aryl-2,2′-anhydro-5-propyl-uridine;5′-O-aryl-2,2′-anhydro-5-benzyl-uridine; and5′-O-aryl-2,2′-anhydro-5-(2-bromovinyl)-uridine; and derivativesthereof. Examples also include 5′-O-aroyl-2,2′-anhydro-5-methyl-uridine;5′-O-aroyl-2,2′-anhydro-5-ethyl-uridine;5′-O-aroyl-2,2′-anhydro-5-propyl-uridine;5′-O-aroyl-2,2′-anhydro-5-benzyl-uridine; and5′-O-aroyl-2,2′-anhydro-5-(2-bromovinyl)-uridine; and derivativesthereof. Compounds of specific interest include5′-O-benzoyl-2,2′-anhydro-5(R⁴)-uridines, such as5′-O-benzoyl-2,2′-anhydro-5-methyl-uridine;5′-O-benzoyl-2,2′-anhydro-5-ethyl-uridine;5′-O-benzoyl-2,2′-anhydro-5-propyl-uridine;5′-O-benzoyl-2,2′-anhydro-5-benzyl-uridine; and5′-O-benzoyl-2,2′-anhydro-5-(2-bromovinyl)-uridine. Stereoisomers ofinterest include the 5′-O-(cyclic hydrocarbyl)-2,2′-anhydropyrimidineswhich are the β-D-isomers. Examples include, but are not limited to:5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)uracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-fluorouracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-trifluoromethyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-ethyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-n-propyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-isopropyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-isobutyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyacyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-propylacyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-(2-bromovinyl)uracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-phenyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-benzyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-benzoyluracil; and5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-(3-benzyloxybenzyl)uracil.

As noted above, also of interest are analogues/derivatives of the abovecompounds.

The 2,2′-anhydropyrimidine and derivatives thereof described above arecommercially available or can be conventionally prepared by techniquesknown to one of skill in the art. For example, representative patentsdescribing various 2,2′-anhydropyrimidine and derivatives, includingintermediates and precursors, analysis, as well as thesynthesis/preparation thereof, include U.S. Pat. Nos. 3,975,367;4,145,531; 4,230,698; 4,247,544; 4,544,740; 4,604,382; 4,613,604;4,681,933; 4,841,039; 4,916,122; 4,987,224; 5,008,384; 5,077,280;5,084,445; 5,141,943; 5,190,926; 5,212,293; 5,278,167; 5,384,396;5,455,339; 5,476,855; 5,596,093; 5,610,292; 5,721,241; 5,723,449;5,739,314; 5,760,202; 5,889,013; 5,861,493; 6,060,592; 6,090,932;6,222,025; 6,369,040; 6,642,367; 6,670,461; 6,867,290; and 7,176,295;the disclosures of which are herein incorporated by reference.

Uridine phosphorylase (UPase) inhibitors also include, but are notlimited to: benzylacyclouridine, benzyloxyacylouridine,aminomethyl-benzylacylouridine,aminomethyl-benzyloxybenzylacyclouridine,hydroxymethyl-benzylacyclouridine, hydroxymethyl-benzyloxybenzylacyclouridine, and the like; derivatives of 5-benzylbarbiturate, such as5-benzyloxybenzyl barbiturate;5-benzyloxybenzyl-1-(1-hydroxy-2-ethoxy)methyl) barbiturate;5-benzyloxybenzylacetyl-1-(1-hydroxy-2-ethoxy) methyl) barbiturate;5-benzyloxybenzyl-1-(1,3-dihydroxy 2-propoxy)methyl barbiturate;5-benzyloxybenzyl-1-(1-hydroxy, 3-amino-2-propoxy)methyl) barbiturate;5-benzyloxybenzyl-1-(2-(3-carboxypropionyloxy)ethoxy) methyl)barbiturate; 5-benzyl-1-(1-hydroxy-2-ethoxy) methyl) barbiturate;5-methoxybenzylacetyl barbiturate;5-benzyl-1-(1,3-dihydroxy-2-propoxy)methyl) barbiturate;5-benzyl-1-(1-hydroxy, 3-amino-2-propoxy)methyl)barbiturate; and5-benzyl-1-(2-(3-carboxypropionyloxy)ethoxy)methyl) barbiturate, and thelike. Upase inhibitors which may be employed in embodiments of theinvention include, but are not limited to, those described in U.S. Pat.Nos. 5,723,449; 5,141,943; 5,077,280; and 4,613,604; the disclosures ofwhich compounds are incorporated herein by reference.

Uridine (UR) Active Agents

As summarized above, in some embodiments the UPase inhibitor isadministered to the subject in combination with a UR active agent (e.g.,uridine (UR), a UR pro-drug or a UR mimetic). Uridine is a nucleosidethat is formed when uracil is attached to a ribose ring (also known as aribofuranose) via a β-N₁-glycosidic bond. Uridine is available inphosphorylated form, i.e., uridine-5′-monophosphate (also known as5′-uridylic acid and UMP), uridine 5′-monophosphate tris salt, uridine5′-monophosphate salt dihydrate, uridine 5′-monophosphate salt solution,uridine 5′-monophosphate salt hydrate, uridine^(13C) ₉, ¹⁵N₂5′-monophosphate sodium salt solution, uridine-¹⁵N₂ 5′-monophosphatesodium salt solution, uridine 5′-monophosphate trisodium salt hydrate,uridine-N₂ 5′-monophosphate sodium salt solution, uridine-5′-diphosphate(UDP), uridine 5′-diphosphate tris salt, uridine 5′-diphosphate saltdihydrate, uridine 5′-diphosphate salt solution, uridine 5′-diphosphatesalt hydrate, uridine^(13C) ₉, ¹⁵N₂ 5′-diphosphate sodium salt solution,uridine-5′-triphosphate (UTP), UTPγS, MRS2498, uridine 5′-triphosphatetris salt, uridine 5′-triphosphate salt dihydrate, uridine5′-triphosphate salt solution, uridine 5′-triphosphate salt hydrate,uridine^(13C) ₉, ¹⁵N₂ 5′-5′-triphosphate sodium salt solution,2-diuridine tetraphosphate, thio-UTP tetrasodium salt, denufosoltetrasodium, or UTP.gamma.S trisodium salt, prodrugs known in the art astriacetyluridine (TAU) or uridine triacetate (PN501), acyl derivativesof uridine such as those described in U.S. Pat. No. 7,582,619 (i.e.,2′,3′,5′-tri-0-pyruvyluridine), 2,2′-anhydro-5-ethyluridine,5-ethyl-2-deoxyuridine, and acyclouridine compounds such as 5-benzylsubstituted acyclouridine congeners including, e.g.,benzylacyclouridine, benzyloxybenzylacyclouridine,aminomethyl-benzylacyclouridine,aminomethylbenzyloxy-benzylacyclouridine,hydroxymethyl-benzyloxybenzylacyclouridine (see also, WO89/09603 andWO91/16315), and in dietary supplements such as Mitocnol and NucleomaxX,derived from sugar cane extract.

UR and sources thereof include, but are not limited to: meat products,such as fish, pig and cow liver and pancreas, and the like; fungirelated products, such as brewer's yeast, beer, mushrooms, and the like;vegetable products, such as sugarcane, tomatoes, oats, algae, broccoliand the like; salts, such as UR phosphates, acylated UR, and the like.UR and sources thereof which may be employed in embodiments of theinvention include, but are not limited to, those described in U.S. Pat.Nos. 9,579,337; 6,316,426; and 5,470,838; the disclosures of whichcompounds are incorporated herein by reference.

UR precursors and sources thereof include, but are not limited to:prodrugs of UR, such as triphenyluridine, orotic acid and the like;prodrugs of uridine 5′-monophosphate, such as mono- and di-alkyl esters,acyloxyalkyl esters, alkoxycarbonylmethyl esters, substituted ethyl andpropyl esters, amidomethyl esters, benzyl esters phenyl esters,phosphonamidates, cyclophosphate esters and the like; UR prodrugscontaining mono-, di- or tri-esters of UR, such as mono-, di-, andtriacetyl UR and the like; UR prodrugs containing mono, di- ortri-phosphates of UR, such as UR monophosphate, UR diphosphate, URtriphosphate and the like; UR homodimers and their esters, such as U-P-Uand the like; heterodimers of dideoxynucleoside compounds and UR orUPase inhibitors, such as AZT-P-U and AZT-P-BAU; etc. Uridine precursorsand sources thereof which may be employed in embodiments of theinvention include, but are not limited to, those described in U.S. Pat.Nos. 5,723,449 and 7,737,128; the disclosures of which compounds areincorporated herein by reference.

Uridine (UR) Processing Modulators

Where desired, a UR processing modulator may also be administered to thesubject in combination with the UPase inhibitor. UR secretion inhibitingcompounds include, but are not limited to: drugs, such as dilazep,hexobendine. UR secretion inhibiting compounds which may be employed inembodiments of the invention include, but are not limited to, thosedescribed in U.S. Pat. Nos. 6,989,376 and 5,567,689; the disclosures ofwhich compounds are incorporated herein by reference.

UR renal transport competitors include, but are not limited to drugs,such as L-uridine, L-2′,3′-dideoxyuridine, D-2′,3′-dideoxyuridine. URrenal transport competitors which may be employed in embodiments of theinvention include, but are not limited to, those described in U.S. Pat.Nos. 6,989,376; 5,723,449 and 5,567,689; the disclosures of whichcompounds are incorporated herein by reference.

Formulations

Also provided are pharmaceutical compositions that find use inembodiments of the invention, e.g., that contain a UPase inhibitorand/or UR active agent, e.g., as described above. The active agent(s)may be present in pharmaceutical compositions, e.g., in the form of apharmaceutically acceptable salt, and can be formulated for oral,topical or parenteral administration for use in the subject methods, asdescribed above. Formulations employed in embodiments of the inventioncan include a single active agent or a combination of active agents. Assuch, embodiments of the invention includes formulations that include asingle active agent, such as a UPase inhibitor or a UR active agent, aswell formulations that include two or more active agents, as where botha UPase inhibitor and a UR active agent are present together in a commonformulation.

By way of illustration, UPase inhibitor and, where desired, a UR activeagent, (separately or in combination) can be admixed with conventionalpharmaceutically acceptable carriers and excipients (e.g., vehicles) andused in the form of aqueous solutions, tablets, capsules, elixirs,suspensions, syrups, wafers, and the like. Such pharmaceuticalcompositions contain, in certain embodiments, from about 0.1% to about90% by weight of the active compound, and more generally from about 1%to about 30% by weight of the active compound. The pharmaceuticalcompositions may contain common carriers and excipients, such as cornstarch or gelatin, lactose, dextrose, sucrose, microcrystallinecellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, andalginic acid. Disintegrators commonly used in the formulations of thisinvention include croscarmellose, microcrystalline cellulose, cornstarch, sodium starch glycolate and alginic acid.

A liquid composition will generally consist of a suspension or solutionof the compound or pharmaceutically acceptable salt in a suitable liquidcarrier(s), for example, ethanol, glycerine, sorbitol, non-aqueoussolvent such as polyethylene glycol, oils or water, with a suspendingagent, preservative, surfactant, wetting agent, flavoring or coloringagent. Alternatively, a liquid formulation can be prepared from areconstitutable powder.

For example, a powder containing active compound, suspending agent,sucrose and a sweetener can be reconstituted with water to form asuspension; and a syrup can be prepared from a powder containing activeingredient, sucrose and a sweetener.

A composition in the form of a tablet can be prepared using any suitablepharmaceutical carrier(s) routinely used for preparing solidcompositions. Examples of such carriers include magnesium stearate,starch, lactose, sucrose, microcrystalline cellulose and binders, forexample, polyvinylpyrrolidone. The tablet can also be provided with acolor film coating, or color included as part of the carrier(s). Inaddition, active compound can be formulated in a controlled releasedosage form as a tablet comprising a hydrophilic or hydrophobic matrix.

A composition in the form of a capsule can be prepared using routineencapsulation procedures, for example, by incorporation of activecompound and excipients into a hard gelatin capsule. Alternatively, asemi-solid matrix of active compound and high molecular weightpolyethylene glycol can be prepared and filled into a hard gelatincapsule; or a solution of active compound in polyethylene glycol or asuspension in edible oil, for example, liquid paraffin or fractionatedcoconut oil can be prepared and filled into a soft gelatin capsule.

Tablet binders that can be included are acacia, methylcellulose, sodiumcarboxymethylcellulose, poly-vinylpyrrolidone (Povidone), hydroxypropylmethylcellulose, sucrose, starch and ethylcellulose. Lubricants that canbe used include magnesium stearate or other metallic stearates, stearicacid, silicone fluid, talc, waxes, oils and colloidal silica.

Flavoring agents such as peppermint, oil of wintergreen, cherryflavoring or the like can also be used. Additionally, it may bedesirable to add a coloring agent to make the dosage form moreattractive in appearance or to help identify the product.

The compounds of the invention and their pharmaceutically acceptablesalts that are active when given parenterally can be formulated forintramuscular, intrathecal, or intravenous administration.

A typical composition for intramuscular or intrathecal administrationwill be of a suspension or solution of active ingredient in an oil, forexample, arachis oil or sesame oil. A typical composition forintravenous or intrathecal administration will be a sterile isotonicaqueous solution containing, for example, active ingredient and dextroseor sodium chloride, or a mixture of dextrose and sodium chloride. Otherexamples are lactated Ringer's injection, lactated Ringer's plusdextrose injection, Normosol-M and dextrose, Isolyte E, acylatedRinger's injection, and the like. Optionally, a co-solvent, for example,polyethylene glycol, a chelating agent, for example, ethylenediaminetetracetic acid, and an anti-oxidant, for example, sodium metabisulphitemay be included in the formulation. Alternatively, the solution can befreeze dried and then reconstituted with a suitable solvent just priorto administration.

The compounds of the invention and their pharmaceutically acceptablesalts which are active on rectal administration can be formulated assuppositories. A typical suppository formulation will generally consistof active ingredient with a binding and/or lubricating agent such as agelatin or cocoa butter or other low melting vegetable or synthetic waxor fat.

The compounds of this invention and their pharmaceutically acceptablesalts which are active on topical administration can be formulated astransdermal compositions or transdermal delivery devices (“patches”).Such compositions include, for example, a backing, active compoundreservoir, a control membrane, liner and contact adhesive. Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds of the present invention in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art. See, eg,U.S. Pat. No. 5,023,252, herein incorporated by reference in itsentirety. Such patches may be constructed for continuous, pulsatile, oron demand delivery of pharmaceutical agents.

In certain embodiments of interest, the UPase inhibitor and UR activeagent are administered as a single pharmaceutical formulation, that, inaddition to the active agent, includes other suitable compounds andcarriers, and may also be used in combination with other active agents.The present invention, therefore, also includes pharmaceuticalcompositions comprising pharmaceutically acceptable excipients. Thepharmaceutically acceptable excipients include, for example, anysuitable vehicles, adjuvants, carriers or diluents, and are readilyavailable to the public. The pharmaceutical compositions of the presentinvention may further contain other active agents that are well known inthe art.

One skilled in the art will appreciate that a variety of suitablemethods of administering a formulation of the present invention to asubject or host, eg, patient, in need thereof, are available, and,although more than one route can be used to administer a particularformulation, a particular route can provide a more immediate and moreeffective reaction than another route. Pharmaceutically acceptableexcipients are also well-known to those who are skilled in the art andare readily available. The choice of excipient will be determined inpart by the particular compound, as well as by the particular methodused to administer the composition. Accordingly, there are a widevariety of suitable formulations of the pharmaceutical composition ofthe present invention. The following methods and excipients are merelyexemplary and are in no way limiting.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachetsor tablets, each containing a predetermined amount of the activeingredient, as solids or granules; (c) suspensions in an appropriateliquid; and (d) suitable emulsions. Tablet forms can include one or moreof lactose, mannitol, corn starch, potato starch, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, diluents, buffering agents, moistening agents, preservatives,flavoring agents, and pharmacologically compatible excipients. Lozengeforms can comprise the active ingredient in a flavor, usually sucroseand acacia or tragacanth, as well as pastilles comprising the activeingredient in an inert base, such as gelatin and glycerin, or sucroseand acacia, emulsions, gels, and the like containing, in addition to theactive ingredient, such excipients as are known in the art.

The subject formulations of the present invention can be made intoaerosol formulations to be administered via inhalation. These aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like. They mayalso be formulated as pharmaceuticals for non-pressured preparationssuch as for use in a nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described. Formulations suitable for topicaladministration may be presented as creams, gels, pastes, or foams,containing, in addition to the active ingredient, and other suchcarriers that are known in the art to be appropriate.

Suppository formulations are also provided by mixing with a variety ofbases such as emulsifying bases or water-soluble bases. Formulationssuitable for vaginal administration may be presented as pessaries,tampons, creams, gels, pastes, foams.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the nature of the deliveryvehicle, and the like. Suitable dosages for a given compound are readilydeterminable by those of skill in the art by a variety of means.

The dose administered to an animal, particularly a human, in the contextof the present invention should be sufficient to cause a prophylactic ortherapeutic response in the animal over a reasonable time frame. Oneskilled in the art will recognize that dosage will depend on a varietyof factors including the strength of the particular compound employed,the condition of the animal, and the body weight of the animal, as wellas the severity of the illness and the stage of the disease. The size ofthe dose will also be determined by the existence, nature, and extent ofany adverse side-effects that might accompany the administration of aparticular compound. Optionally, the pharmaceutical composition maycontain other pharmaceutically acceptable components, such as buffers,surfactants, antioxidants, viscosity modifying agents, preservatives andthe like. Each of these components is well-known in the art. Forexample, see U.S. Pat. No. 5,985,310, the disclosure of which is hereinincorporated by reference.

Other components suitable for use in the formulations of the presentinvention can be found in Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, Pa., 17th ed. (1985). In anembodiment, the aqueous solution of cyclodextrin also contains dextrose,eg, about 5% dextrose.

Utility

The subject methods find use in the treatment liver diseases. While thetarget liver disease may vary, in some instances the liver diseases arecharacterized by the presence of feature fibrosis, or the accumulationof extracellular matrix molecules that make up scar tissue as the toxicendpoint, as well as other diseases such as, inter alia, pulmonaryfibrosis, renal fibrosis, systemic sclerosis (SSc), sclerodermatousgraft vs. host disease, radiation-induced fibrosis and cardiac fibrosis.

In some instances, the liver disease is a fatty liver disorder. Fattyliver disorders, also known as fatty liver or fatty liver disease (FLD),relates to a condition where large vacuoles of triglyceride fataccumulate in liver cells via the process of steatosis, or abnormalretention of lipids within a cell. Despite having multiple causes, fattyliver is considered a single disease that occurs frequently in subjectswith excessive alcohol intake and those who are obese (with or withouteffects of insulin resistance). The condition is also associated withother diseases that influence fat metabolism. FLD may be categorizedinto two separate conditions: alcoholic FLD and non-alcoholic FLD. Bothconditions show micro-vesicular and macro-vesicular fatty changes atdifferent stages of the disease. Accumulation of fat may also beaccompanied by a progressive inflammation of the liver (hepatitis),called steatohepatitis. Fatty liver is also known in the art asalcoholic steatosis and non-alcoholic fatty liver disease (NAFLD), andthe more severe forms as alcoholic steatohepatitis (part of alcoholicliver disease) and non-alcoholic steatohepatitis (NASH). Nonalcoholicfatty liver disease-associated cirrhosis is the most severe form of thedisease and is characterized by liver inflammation that leads toscarring of the liver tissue, ultimately resulting in liver failure. Insome instances, the liver condition is NAFLD, NASH or DILI.

By treatment, is meant that at least an amelioration of the symptomsassociated with the condition afflicting the host is achieved, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g., a symptom associated with thecondition being treated or a side effect resulting from administrationof a drug. As such, treatment also includes situations where thepathological condition, or at least symptoms associated therewith, arecompletely inhibited, e.g., prevented from happening, or stopped, e.g.,terminated, such that the host no longer suffers from the condition, orat least the symptoms that characterize the condition. Treating alsoinclude prophylactically treating the subject, such that the livercondition does not occur in the subject. As such, treating includespreventing the occurrence of the liver condition in the subject.

A variety of subjects are treatable according to the subject methods.Generally such hosts are “mammals” or “mammalian,” where these terms areused broadly to describe organisms which are within the class mammalia,including the orders carnivore (eg, dogs and cats), rodentia (eg, mice,guinea pigs, and rats), and primates (eg, humans, chimpanzees, andmonkeys). In many embodiments, the subjects will be humans.

In certain embodiments, the subjects will be subjects that have beendiagnosed for and are, therefore, in need of administration of theactive agent. In certain embodiments, the methods may include diagnosingthe subject for the presence of the disease condition to be treated byadministration of the active agent.

Where the liver disease is DILI, the methods of the invention may beemployed in combination with the therapeutic regimen that is suspectedto cause the DILI, e.g., to treat DILI that has already occurred or toprophylactically treat DILI. Where the compounds of the invention areadministered in conjunction with other therapies, dosages of theco-administered compounds will of course vary depending on the type ofco-drug employed, on the specific drug employed, on the condition beingtreated and so forth. As used herein, the terms “combination treatment”,“combination therapy”, “combined treatment” or “combinatorialtreatment”, used interchangeably, refer to a treatment of an individualwith at least two different therapeutic agents. The terms“co-administration” or “combined administration” or the like as utilizedherein are meant to encompass administration of the selected therapeuticagents to a single patient, and are intended to include treatmentregimens in which the agents are not necessarily administered by thesame route of administration or at the same time. The term“pharmaceutical combination” means a product that results from themixing or combining of more than one active ingredient and includes bothfixed and non-fixed combinations of the active ingredients. A “fixedcombination” means that the active ingredients, e.g. a compound asdisclosed herein and one or more additional therapeutic agents, are bothadministered to a patient simultaneously in the form of a single entityor dosage. A “non-fixed combination” means that the active ingredients,e.g. a compound as disclosed herein and one or more additionaltherapeutic agents, are both administered to a patient as separateentities either simultaneously, concurrently or sequentially with nospecific time limits, wherein such administration providestherapeutically effective levels of the 2 compounds in the body of thepatient. The latter also applies to cocktail therapy, e.g. theadministration of 3 or more active ingredients. As used herein, methodsof the invention may employed in combination with one or more additionaltherapeutic agents, such as, without limitation, agents for pulmonaryhypertension, such as ambrisentan, bosentan, treprostinil, sildenafil,epoprostenol, treprostinil, iloprost, aldosterone receptor antagonistslike spironolactone and eplerenone, angiotensin-converting enzymeinhibitors such as trandolapril, fosinopril, enalapril, captopril,ramipril, moexipril, lisinopril, quinapril, benazepril, and perindopril,angiotensin II inhibitors such as eprosartan, olmesmian, telmismian,losartan, valsmian, candesartan, and irbesmian, anti-anginal agents likenitroglycerin, isosorbide mononitrate, and isosorbide dinitrate,anti-arrhythmic agents including moricizine, quinidine, diisopyramide,phenyloin, propafenone, flecaimide, mexiletine, lidocaine, procainamide,propranolol, acebutolol, amiodarone, dofetilide, dronedarone, sotalol,ibutilide, diltiazem, verapamil, nifedipine, nimodipine, felodipine,nicardipine, clevidipine, isradipine, bepridil, nisoldipine, adenosine,and digoxin, P-adrenergic receptor antagonists like betaxolol,bisoprolol, metoprolol, atenolol, nebivolol, nadolol, carvedilol,labetalol, timolol, carteolol, penbutolol, pindolol, and esmolol,anti-diabetic agents including secretagogues such as sulfonylurea,tolbutamide, acetohexamide, tolazamide, chlorpropamide, glipizide,glyburide, glimepiride, glibenclamide, gliclazide, meglitinide such asnateglinide, senaglinide, repaglinide, insulin sensitizers such asbiguanides, metformin, thiazolidinediones such as rosiglitazone,isaglitazone, darglitazone, englitazone, and pioglitazone, a-glucosidaseinhibitors such as miglitol, voglibose, emiglitate, and acarbose,glucagon-like peptide analogs and agonists such as exenatide,liraglutide, and taspglutide, dipeptidyl peptidase-4 inhibitors likevildagliptin, sitagliptin, and saxagliptin, amylin analogs such aspramlintide, ligands or agonists of peroxisome proliferator activatedreceptor (PPAR)-.alpha., .beta., .delta., and .gamma.cholesterol-lowering agents such as hydroxymethylglutaryl-Coenzyme A(HMG-CoA) reductase inhibitors like statins, such as, e.g.,atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin,rosuvastatin, and simvastatin, agonists of retinoid X receptors (RXR)such as, e.g., ALRT-268, LG-1268, or LG-1069, glucokinase activators,inhibitors of hepatic enzymes involved in stimulation of gluconeogenesisand/or glycogenolysis, diuretics such as acetazolamide,dichlorphenamide, methazolamide, torsemide, furosemide, bumetanide,ethacrynic acid, amiloride, triamterene, indapamide, metolazone,methylclothiazide, hydrochlorothiazide, chlorothiazide, metolazone,bendroflumethiazide, polythiazide, and chlorthalidone, vasodilators likealprostadil, hydralazine, minoxidil, nesiritide, and nitroprusside, andother anti-lipidemic agents like cholestyramine, colestipol, clofibrate,gemfibrozil, probucol or dextrothyroxine.

Kits & Systems

Also provided are kits and systems that find use in practicing thesubject methods, eg, as described above. For example, kits and systemsfor practicing the subject methods may include one or morepharmaceutical formulations, which include the UPase inhibitor and, insome embodiments a UR active agent. As such, in certain embodiments thekits may include a single pharmaceutical composition, present as one ormore unit dosages, where the composition includes both UPase inhibitorand a UR active agent. In yet other embodiments, the kits may includetwo or more separate pharmaceutical compositions, each containing aUPase inhibitor and, optionally, a UR active agent.

In addition to the above components, the subject kits may furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, eg, a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium, eg,diskette, CD, etc., on which the information has been recorded. Yetanother means that may be present is a website address which may be usedvia the internet to access the information at a removed site. Anyconvenient means may be present in the kits. For example, a kitaccording to one embodiment includes as a first component (a)instructions for using a plasma UR level modulator, and as a secondcomponent (b) a pharmaceutical composition comprising a uridine, an URprodrug, or an UR mimetic.

Kits of specific interest are those that include a 2,2′-anhydropyrimidine pharmaceutical composition of the invention andsuitable for practicing the subject methods of the invention, such asfor mitigating serious liver conditions.

The term “system” as employed herein refers to a collection of a UPaseinhibitor, and, optionally a UR active agent present in a single ordisparate composition, that are brought together for the purpose ofpracticing the subject methods. For example, separately obtained UPaseinhibitor and UR active agent dosage forms brought together andco-administered to a subject, according to the present invention, are asystem according to the present invention.

The following examples further illustrate the present invention butshould not be construed in any way as limiting its scope.

EXAMPLES

I. Increase in UR with Increasing Compound I Concentrations.

Because UR is cleared so rapidly, elimination t_(1/2) only a fewminutes,⁴⁶ and the elimination t_(1/2) of Compound 1 in mice is only 1-2hours, it is very challenging to measure UR concentrations elevationspost discrete doses of Compound I, such as used for ip dosing. For thisreason continuous infusion of compound I (authentic TK-112690, BatchTCY90108) to BDF-1 ♂ mice were administered via a sc implanted osmoticpumps and the UR plasma concentration measured. ⁴⁶ Deng Y, Wang Z V,Gordillo R, An Y, Zhang C, Liang Q, Yoshino J, Cautivo K M, De BrabanderJ, Elmquist J K, Horton J D, Hill J A, Klein S, Scherer P E. Anadipo-biliary-uridine axis that regulates energy homeostasis. Science2017, 17; 355(6330)

Solutions of Compound I were prepared at a concentration of 500 mg/mL insterile PBS. Osmotic pumps (ALZET® micro-osmotic pump 2001D and 1003D,Alza Co) were loaded with 200 μL (2001D osmotic pump) and/or 100 μL(1003D osmotic pump) of TK-112690 solution.

BDF-1 male mice (n=6) were treated with a constant-rate infusion of 667,833 or 3000 mg/kg/day doses of Compound I delivered via subcutaneouslyimplanted osmotic pumps. Animals were anesthetized with 100 mg/kgketamine prior to pump implantation. Surgical scissors were used to makean approximately 1 cm incision on the dorsal surface near the shoulderblade of animals. A hemostat was used to carve out a subcutaneous tunneltoward the anterior end of animal. Osmotic pumps were placed inside thesubcutaneous tunnel. Incision was sealed with wound clips.

Blood collections were performed on animals anesthetized with ketamine(ip 100 mg/kg). Blood samples from animals treated with a constant-rateinfusion of TK-112690 were collected at 72 hours for 667 mg/kg/day and833 mg/kg/day and 24 hours for 3000 mg/kg/day after pump implantation.Whole blood (˜0.8 mL) was drawn through the retro-orbital sinus using aheparin coated micro-hematocrit tube and collected into an EDTAmicrotainer tube. Blood samples were transferred into fresh 1.5 mLmicrocentrifuge tubes, and centrifuged for 10 minutes at 14,000×g usingan Eppendorf Minispin Plus stored in a 4° C. refrigerator. Exactly 0.4mL of plasma was transferred into fresh microcentrifuge tubes containing2 μL of 10 mM 5-FU and vortexed at highest setting for approximately 5seconds. The final 50 μM concentration of 5-FU served as an internalstandard. Animals were sacrificed by cervical dislocation and properlydisposed.

Blood samples from animals treated with a constant-rate infusion ofcompound I were collected at 72 hours for 667 mg/kg/day and 833mg/kg/day and 24 hours for 3000 mg/kg/day after pump implantation. Wholeblood (˜0.8 mL) was drawn through the retro-orbital sinus using aheparin coated micro-hematocrit tube and collected into an EDTAmicrotainer tube. Blood samples were transferred into fresh 1.5 mLmicrocentrifuge tubes, and centrifuged for 10 minutes at 14,000×g usingan Eppendorf Minispin Plus stored in a 4° C. refrigerator. Exactly 0.4mL of plasma was transferred into fresh microcentrifuge tubes containing2 μL of 10 mM 5-FU and vortexed at highest setting for approximately 5seconds. The final 50 μM concentration of 5-FU served as an internalstandard. Animals were sacrificed by cervical dislocation and properlydisposed.

A solid-phase extraction (SPE) of analytes (UR, Compound I and 5-FU)from plasma was conducted before HPLC analysis. Supelco C8 SPE columnswere used for extraction process. All solutions were pushed through SPEcolumns using positive pressure generated from a Vacuum-Pressure Pump(Barnant Company Model 400-1901). The flow rate through the SPE columnwas approximately 2 drops per second. Pre-washing of SPE columns wasdone with a total of 2.4 mL of sterile PBS (room temp; pH=7.4). Exactly0.6 mL PBS was added to the SPE column four separate times and pushedthrough the column. Immediately after pre-wash, all 0.4 mL of the plasmasample (with added 5-FU internal standard) was transferred onto thecolumn and pushed through the column. Analytes were disassociated fromSPE column by pushing through exactly 0.5 mL of 5 M NaCl (room temp; pH5). Eluted samples were collected into fresh 1.5 mL micro-centrifugetubes. Samples were transferred into fresh HPLC vials and analyzed.

HPLC analysis was done at room temperature (RT) using a ThermoFinniganSpectra System equipped with degasser, pump, autosampler and UVdetector. Chromatograms were constructed from a chart recorder equippedwith a pen. Analytes were separated using a Phenomenex C18 Reverse-Phasecolumn (250×4.6 mm). Two separate mobile phase gradients were employedfor the HPLC analysis: (1) 5% methanol in nano water with 0.1% formicacid (2) 5% methanol in acetonitrile with 0.1% formic acid (flowrate=0.5 mL per minute). HPLC responses for compound I and UR weredivided by the 5-FU response. Calibration curves were used to convertthese ratios into concentrations of Compound 1.

A regression analysis (UR concentration vs. Compound 1 concentration)for data from the study is provided in FIG. 1. Higher concentrations ofCompound 1 are seen to be associated with higher levels of UR.

II. Methionine-Choline Diet (MCD) Model of NASH.

Mice fed an MCD diet is a standard model of diet induced NASH.^(47,48)All animals were housed in ventilated standard housing cages throughoutthe experimental phase. Tap water was provided ad libitum to allanimals. Male, 8-week old, C57BL/6 mice from Charles River wereacclimated for 3 days maintained on a standard chow diet and grouphoused in hepa-filtered cages (5 animals per cage) on a normal 12 hourslight cycle (at 8 am to 8 pm lights off). The temperature and humiditywere 22±2° C. and 50±10%, respectively. Cages litters were changed oncea week. ⁴⁷ Reid D T, Reyes J L, McDonald B A, Vo T, Reimer R A, EksteenB. Kupffer Cells Undergo Fundamental Changes during the Development ofExperimental NASH and Are Critical in Initiating Liver Damage andInflammation. PLoS One. 2016, 25; 11:e0159524.⁴⁸ Ramadori P, WeiskirchenR, Trebicka J, Streetz K. Mouse models of metabolic liver injury. LabAnim. 2015; 49(1 Suppl):47-58.

Following acclimation, the animals were randomized into homogenoustreatment groups according to their body weight, and fed ad libitum adiet deficient in methionine and choline: 4.2 kcal/g; MP Biomedicals,Solon, Ohio). Food and water intake were measured 3 times per week (atthe same time as body weight measurements). After the acclimationperiod, the 24 mice (n=6/treatment group) were weighed 3 times per weekuntil sacrifice.

The 4 treatment group were:

-   -   Group 1: Vehicle+MCD    -   Group 2: MCD+200 mg/kg UR    -   Group 3: MCD+60 mg/kg Compound I    -   Group 4: MCD+60 mg/kg Compound 1 plus 200 mg/kg UR 30 minutes        post Compound 1

All Compound 1 and UR doses (mg/kg in 10 mL/kg of vehicle) were ip, bidat least 8 hours apart, for 28 days. Vehicle=PBS. Vehicle, UR andCompound 1±UR will be dosed daily starting 2 days prior to placing theanimals on a MCD diet. Mice were on the MCD diet for 26 days.

After 26 days on MCD, the animals were sacrificed ˜2 hours after thelast dose and non-fasted glucose was measured along with a plasma lipidpanel, TNF-α, ALT and AST. Total body weight was determined weekly.Total liver was weighed and the medial lobe excised, formalin fixed andstained with Oil Red O to evaluate lipid content. An intensity score forthe fixed tissue slides measured by an independent histopathologist wasalso determined.

As expected,⁴⁹ body weight decreased for all treatment groups byapproximately 20% (FIG. 2, Data presented as mean+/−SEM.). There was nodifference in body weight between groups. There was also no differencewith Groups 2, 3, and 4 with respect to total triglycerides,cholesterol, AST or ALT. There was a statistical difference betweenGroup 1 and the group 4 values for HDL-cholesterol (FIG. 3) (Serum HDLcholesterol concentrations measured at the end of the MCD study. Dataare means+/−SEM. TK-90 is Compound 1, also known as TK-112690). Mostimportant are the findings for fibrosis (FIG. 4)((A) Representative H&Eimages of liver sections from each experimental group. (B) Fibrosisscoring of the images shown in (A). TK-90 is Compound 1, also known asTK-112690. Data are means+/−SEM.) The reason for the importance of thedata is that fibrosis is the pathologic endpoint for NASH. For the datain FIG. 4, Groups 2, 3, and 4 were all statistically different from theGroup 1 control group. ⁴⁹ 10. Veteläinen R, van Viet A, van Gulik T M.Essential pathogenic and metabolic differences in steatosis induced bycholine or methione-choline deficient diets in a rat model. JGastroenterol Hepatol. 2007; 22:1526-33.

Notwithstanding the appended claims, the disclosure is also defined bythe following clauses:

1. A method of treating a subject for a liver condition, the methodcomprising:

administering to the subject an effective amount of a2,2′-anhydropyrimidine or derivative thereof to treat the subject forthe liver condition.

2. The method according to Clause 1, wherein the 2,2′-anhydropyrimidineor derivative thereof is a compound of formula (I):

or the pharmaceutically acceptable salts, solvates, hydrates, andprodrug forms thereof, and stereoisomers thereof;

wherein:

each R¹, R², R³ and R⁴ is independently selected from the groupconsisting of hydrogen, substituted or unsubstituted heteroatom,substituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,substituted or unsubstituted aralkyl, carbohydrate, nucleic acid, aminoacid, peptide, dye, fluorophore and polypeptide.

3. The method according to Clause 2, wherein each R¹, R², R³ and R⁴ isindependently selected from the group consisting of hydrogen, hydroxyl,sulfhydryl, amino, hydroxymethyl, methoxy, halogen, pseudohalogen, and asubstituted or unsubstituted lower hydrocarbon containing 1 to 20carbons.4. The method according to Clause 2, wherein the lower hydrocarbon isselected from the group consisting of alkyl, alkenyl, alkanoyl, aryl,aroyl, aralkyl and alkylamino, and esters thereof.5. The method according to Clause 2, wherein R¹ is hydrogen, fluorine,methyl, ethyl, propyl, benzyl, or 2-bromovinyl; R² is hydrogen, hydroxylfluorine, methyl, ethyl, propyl, benzyl, benzoyl, benzoyloxy, or2-bromovinyl; and each R³ and R⁴ is independently selected from thegroup consisting of hydroxyl and benzoyloxy.6. The method according to Clause 5, wherein R¹ is hydrogen or methyl;R² is hydrogen; and each R³ and R⁴ is independently selected from thegroup consisting of hydroxyl and benzoyloxy.7. The method according to Clause 1, wherein the 2,2′-anhydropyrimidineor derivative thereof is selected from the group consisting of:2,2′-anhydro-5-methyluridine; 3′-O-benzoyl-2,2′-anhydrouridine;3′-O-benzoyl-2,2′-anhydro-5-methyluridine;5′-O-benzoyl-2,2′-anhydrouridine; and5′-O-benzoyl-2,2′-anhydro-5-methyluridine.8. The method according to Clause 7, wherein the 2,2′-anhydropyrimidineor derivative thereof is 2,2′-anhydro-5-methyluridine.9. The method according to Clause 7, wherein the 2,2′-anhydropyrimidineor derivative thereof is 3′-O-benzoyl-2,2′-anhydro-5-methyluridine.10. The method according to Clause 7, wherein the 2,2′-anhydropyrimidineor derivative thereof is 5′-O-benzoyl-2,2′-anhydro-5-methyluridine.11. The method according to Clause 1, wherein the 2,2′-anhydropyrimidineor derivative thereof comprises a stereoisomer.12. The method according to Clause 11, wherein the stereoisomer isselected from the group consisting of2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyluracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-uracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-uracil; and5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyluracil.13. The method according to any of the preceding clauses, wherein theliver condition is selected from the group consisting of NAFLD, NASH andDILI.14. The method according to any of the preceding clauses, wherein thetreatment is prophylactic.15. The method according to Clause 14, wherein the liver condition isDILI.16. The method according to any of Clauses 1 to 13, wherein the subjectsuffers from the liver condition.17. A method of treat a subject for a liver condition, the methodcomprising: administering to the subject an effective amount of a2,2′-anhydropyrimidine or derivative thereof in combination with auridine (UR) active agent to treat the subject for the liver condition.18. The method according to Clause 17, wherein the2,2′-anhydropyrimidine or derivative thereof is a compound of formula(I):

or the pharmaceutically acceptable salts, solvates, hydrates, andprodrug forms thereof, and stereoisomers thereof;

wherein:

each R¹, R², R³ and R⁴ is independently selected from the groupconsisting of hydrogen, substituted or unsubstituted heteroatom,substituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,substituted or unsubstituted aralkyl, carbohydrate, nucleic acid, aminoacid, peptide, dye, fluorophore and polypeptide.

19. The method according to Clause 18, wherein each R¹, R², R³ and R⁴ isindependently selected from the group consisting of hydrogen, hydroxyl,sulfhydryl, amino, hydroxymethyl, methoxy, halogen, pseudohalogen, and asubstituted or unsubstituted lower hydrocarbon containing 1 to 20carbons.20. The method according to Clause 18, wherein the lower hydrocarbon isselected from the group consisting of alkyl, alkenyl, alkanoyl, aryl,aroyl, aralkyl and alkylamino, and esters thereof.21. The method according to Clause 18, wherein R¹ is hydrogen, fluorine,methyl, ethyl, propyl, benzyl, or 2-bromovinyl; R² is hydrogen, hydroxylfluorine, methyl, ethyl, propyl, benzyl, benzoyl, benzoyloxy, or2-bromovinyl; and each R³ and R⁴ is independently selected from thegroup consisting of hydroxyl and benzoyloxy.22. The method according to Clause 21, wherein R¹ is hydrogen or methyl;R² is hydrogen; and each R³ and R⁴ is independently selected from thegroup consisting of hydroxyl and benzoyloxy.23. The method according to Clause 18, wherein the2,2′-anhydropyrimidine or derivative thereof is selected from the groupconsisting of: 2,2′-anhydro-5-methyluridine;3′-O-benzoyl-2,2′-anhydrouridine;3′-O-benzoyl-2,2′-anhydro-5-methyluridine;5′-O-benzoyl-2,2′-anhydrouridine; and5′-O-benzoyl-2,2′-anhydro-5-methyluridine.24. The method according to Clause 23, wherein the2,2′-anhydropyrimidine or derivative thereof is2,2′-anhydro-5-methyluridine.25. The method according to Clause 23, wherein the2,2′-anhydropyrimidine or derivative thereof is3′-O-benzoyl-2,2′-anhydro-5-methyluridine.26. The method according to Clause 23, wherein the2,2′-anhydropyrimidine or derivative thereof is5′-O-benzoyl-2,2′-anhydro-5-methyluridine.27. The method according to Clause 18, wherein the2,2′-anhydropyrimidine or derivative thereof comprises a stereoisomer.28. The method according to Clause 27, wherein the stereoisomer isselected from the group consisting of2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyluracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-uracil;3′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyluracil;5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-uracil; and5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyluracil.29. The method according to any of Clauses 17 to 28, wherein the livercondition is selected from the group consisting of NAFLD, NASH and DILI.30. The method according to any of Clauses 17 to 29, wherein thetreatment is prophylactic.31. The method according to Clause 30, wherein the liver condition isDILI.32. The method according to any of Clauses 17 to 29, wherein the subjectsuffers from the liver condition.

In at least some of the previously described embodiments, one or moreelements used in an embodiment can interchangeably be used in anotherembodiment unless such a replacement is not technically feasible. Itwill be appreciated by those skilled in the art that various otheromissions, additions and modifications may be made to the methods andstructures described above without departing from the scope of theclaimed subject matter. All such modifications and changes are intendedto fall within the scope of the subject matter, as defined by theappended claims.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible sub-rangesand combinations of sub-ranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into sub-ranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 articles refers to groupshaving 1, 2, or 3 articles. Similarly, a group having 1-5 articlesrefers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. Moreover, nothing disclosedherein is intended to be dedicated to the public regardless of whethersuch disclosure is explicitly recited in the claims.

The scope of the present invention, therefore, is not intended to belimited to the exemplary embodiments shown and described herein. Rather,the scope and spirit of present invention is embodied by the appendedclaims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) isexpressly defined as being invoked for a limitation in the claim onlywhen the exact phrase “means for” or the exact phrase “step for” isrecited at the beginning of such limitation in the claim; if such exactphrase is not used in a limitation in the claim, then 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is not invoked.

What is claimed is:
 1. A method of treating a subject for a liver condition selected from the group consisting of NAFLD and NASH, the method comprising: administering to the subject an effective amount of a 2,2′-anhydropyrimidine or derivative thereof to ameliorate fibrosis to treat the subject for the liver condition, wherein the subject suffers from the liver condition.
 2. The method according to claim 1, wherein the 2,2-anhydropyrimidine or derivative thereof is a compound of formula (I):

or the pharmaceutically acceptable salts, solvates, hydrates, and prodrug forms thereof, and stereoisomers thereof; wherein: each R¹, R², R³ and R⁴ is independently selected from the group consisting of hydrogen, substituted or unsubstituted heteroatom, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, carbohydrate, nucleic acid, amino acid, peptide, dye, fluorophore and polypeptide.
 3. The method according to claim 2, wherein each R¹, R², R³ and R⁴ is independently selected from the group consisting of hydrogen, hydroxyl, sulfhydryl, amino, hydroxymethyl, methoxy, halogen, pseudohalogen, and a substituted or unsubstituted lower hydrocarbon containing 1 to 20 carbons.
 4. The method according to claim 2, wherein the lower hydrocarbon is selected from the group consisting of alkyl, alkenyl, alkanoyl, aryl, aroyl, aralkyl and alkylamino, and esters thereof.
 5. The method according to claim 2, wherein R¹ is hydrogen, fluorine, methyl, ethyl, propyl, benzyl, or 2-bromovinyl; R² is hydrogen, hydroxyl fluorine, methyl, ethyl, propyl, benzyl, benzoyl, benzoyloxy, or 2-bromovinyl; and each R³ and R⁴ is independently selected from the group consisting of hydroxyl and benzoyloxy.
 6. The method according to claim 5, wherein R¹ is hydrogen or methyl; R² is hydrogen; and each R³ and R⁴ is independently selected from the group consisting of hydroxyl and benzoyloxy.
 7. The method according to claim 1, wherein the 2,2′-anhydropyrimidine or derivative thereof is selected from the group consisting of: 2,2′-anhydro-5-methyluridine; 3′-O-benzoyl-2,2′-anhydrouridine; 3*-O-benzoyl-2,2′-anhydro-5-methyluridine; 5′-O-benzoyl-2,2′-anhydrouridine; and 5′-O-benzoyl-2,2′-anhydro-5-methyluridine.
 8. The method according to claim 7, wherein the 2,2′-anhydropyrimidine or derivative thereof is 2,2′-anhydro-5-methyluridine.
 9. The method according to claim 7, wherein the 2,2′-anhydropyrimidine or derivative thereof is 3′-O-benzoyl-2,2′-anhydro-5-methyluridine.
 10. The method according to claim 7, wherein the 2,2′-anhydropyrimidine or derivative thereof is 5′-O-benzoyl-2,2′-anhydro-5-methyluridine.
 11. The method according to claim 1, wherein the 2,2′-anhydropyrimidine or derivative thereof comprises a stereoisomer.
 12. The method according to claim 11, wherein the stereoisomer is selected from the group consisting of 2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyluracil; 3-O-benzoyl-2,2-anhydro-1-(β-D-arabinofuranosyl)-uracil; 3-O-benzoyl-2,2-anhydro-1-(β-D-arabinofuranosyl)-5-methyluracil; 5-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-uracil; and 5′-O-benzoyl-2,2′-anhydro-1-(β-D-arabinofuranosyl)-5-methyluracil.
 13. The method according to claim 1, wherein the liver condition is NASH.
 14. The method according to claim 1, wherein the liver condition is NAFLD.
 15. A method of treating a subject for a liver condition selected from the group consisting of NAFLD and NASH, the method comprising: administering to the subject an effective amount of a 2,2′-anhydropyrimidine or derivative thereof in combination with a uridine (UR) active agent to ameliorate fibrosis to treat the subject for the liver condition, wherein the subject suffers from the liver condition.
 16. The method according to claim 15, wherein the 2,2′-anhydropyrimidine or derivative thereof is a compound of formula (I):

or the pharmaceutically acceptable salts, solvates, hydrates, and prodrug forms thereof, and stereoisomers thereof; wherein: each R¹, R², R³ and R⁴ is independently selected from the group consisting of hydrogen, substituted or unsubstituted heteroatom, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, carbohydrate, nucleic acid, amino acid, peptide, dye, fluorophore and polypeptide.
 17. The method according to claim 15, wherein the liver condition is NASH.
 18. The method according to claim 15, wherein the liver condition is NAFLD. 